RNA Gel Blot Hybridization Research Articles

Physcomitrium patens pentatricopeptide repeat protein PpPPR_32 is involved in the accumulation of psaC mRNA encoding the iron sulfur protein of photosystem I.

Pentatricopeptide repeat (PPR) proteins are involved in RNA metabolism and also play a role in posttranscriptional regulation during plant organellar gene expression. Although a hundred of PPR proteins exist in the moss Physcomitrium patens, their functions are not fully understood. Here, we report the function of P-class PPR protein PpPPR_32 in P. patens. A transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpPPR_32 functions as a chloroplast-targeting transit peptide, indicating that PpPPR_32 is localized in chloroplasts. PpPPR_32 knockout mutants grew autotrophically but with reduced protonema growth and the poor formation of photosystem I (PSI) complexes. Quantitative real-time reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in the transcript level of the psaC gene encoding the iron sulfur protein of PSI but no alteration to the transcript levels of other PSI genes. This suggests that PpPPR_32 is specifically involved in the expression level of the psaC gene. Our results indicate that PpPPR_32 is essential for the accumulation of psaC transcript and PSI complexes.

Moss PPR-SMR protein PpPPR_64 influences the expression of a psaA-psaB-rps14 gene cluster and processing of the 23S-4.5S rRNA precursor in chloroplasts.

Moss PPR-SMR protein PpPPR_64 is a pTAC2 homolog but is functionally distinct from pTAC2. PpPPR_64 is required for psaA gene expression and its function may have evolved in mosses. The pentatricopeptide repeat (PPR) proteins are key regulatory factors responsible for the control of plant organellar gene expression. A small subset of PPR proteins possess a C-terminal small MutS-related (SMR) domain and have diverse roles in plant organellar biogenesis. However, the function of PPR-SMR proteins is not fully understood. Here, we report the function of PPR-SMR protein PpPPR_64 in the moss Physcomitrium patens. Phylogenetic analysis indicated that PpPPR_64 belongs to the same clade as the Arabidopsis PPR-SMR protein pTAC2. PpPPR_64 knockout (KO) mutants grew autotrophically but with reduced protonemata growth and the poor formation of photosystems' antenna complexes. Quantitative reverse transcription-polymerase chain reaction and RNA gel blot hybridization analyses revealed a significant reduction in transcript levels of the psaA-psaB-rps14 gene cluster but no alteration to transcript levels of most photosynthesis- and non-photosynthesis-related genes. In addition, RNA processing of 23S-4.5S rRNA precursor was impaired in the PpPPR_64 KO mutants. This suggests that PpPPR_64 is specifically involved in the expression level of the psaA-psaB-rps14 gene and in processing of the 23S-4.5S rRNA precursor. Our results indicate that PpPPR_64 is functionally distinct from pTAC2 and is a novel PPR-SMR protein required for proper chloroplast biogenesis in P. patens.

The Chloroplast RNA Binding Protein CP31A Has a Preference for mRNAs Encoding the Subunits of the Chloroplast NAD(P)H Dehydrogenase Complex and Is Required for Their Accumulation.

Chloroplast RNA processing requires a large number of nuclear-encoded RNA binding proteins (RBPs) that are imported post-translationally into the organelle. Most of these RBPs are highly specific for one or few target RNAs. By contrast, members of the chloroplast ribonucleoprotein family (cpRNPs) have a wider RNA target range. We here present a quantitative analysis of RNA targets of the cpRNP CP31A using digestion-optimized RNA co-immunoprecipitation with deep sequencing (DO-RIP-seq). This identifies the mRNAs coding for subunits of the chloroplast NAD(P)H dehydrogenase (NDH) complex as main targets for CP31A. We demonstrate using whole-genome gene expression analysis and targeted RNA gel blot hybridization that the ndh mRNAs are all down-regulated in cp31a mutants. This diminishes the activity of the NDH complex. Our findings demonstrate how a chloroplast RNA binding protein can combine functionally related RNAs into one post-transcriptional operon.

Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23.

Key messageCitrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle.The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

Stable Membrane-Association of mRNAs in Etiolated, Greening and Mature Plastids.

Chloroplast genes are transcribed as polycistronic precursor RNAs that give rise to a multitude of processing products down to monocistronic forms. Translation of these mRNAs is realized by bacterial type 70S ribosomes. A larger fraction of these ribosomes is attached to chloroplast membranes. This study analyzed transcriptome-wide distribution of plastid mRNAs between soluble and membrane fractions of purified plastids using microarray analyses and validating RNA gel blot hybridizations. To determine the impact of light on mRNA localization, we used etioplasts, greening plastids and mature chloroplasts from Zea mays as a source for membrane and soluble extracts. The results show that the three plastid types display an almost identical distribution of RNAs between the two organellar fractions, which is confirmed by quantitative RNA gel blot analyses. Furthermore, they reveal that different RNAs processed from polycistronic precursors show transcript-autonomous distribution between stroma and membrane fractions. Disruption of ribosomes leads to release of mRNAs from membranes, demonstrating that attachment is likely a direct consequence of translation. We conclude that plastid mRNA distribution is a stable feature of different plastid types, setting up rapid chloroplast translation in any plastid type.

Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: implications for detection

Natural populations of peach latent mosaic viroid (PLMVd) are complex mixtures of variants. During routine testing, TaqMan rtRT-PCR and RNA gel-blot hybridization produced discordant results with some PLMVd isolates. Analysis of the corresponding populations showed that they were exclusively composed of variants (of class II) with a structural domain different from that of the reference and many other variants (of class I) targeted by the TaqMan rtRT-PCR probe. Bioassays in peach revealed that a representative PLMVd variant of class II replicated without symptoms, generated a progeny with low nucleotide diversity, and, intriguingly, outcompeted a representative symptomatic variant of class I when co-inoculated in equimolecular amounts. A number of informative positions associated with the higher fitness of variants of class II have been identified, and novel sets of primers and probes for universal or specific TaqMan rtRT-PCR detection of PLMVd variants have been designed and tested.

Characterization of the stage dependency of high temperature on green ripening reveals a distinct chlorophyll degradation regulation in banana fruit

Bananas (Musa, AAA group) develop a bright yellow appearance when ripen at temperatures between 18°C and 24°C. At high ambient temperatures, the fruit display green-ripe and become un-acceptable in the market. To understand the mechanisms that govern the color changing during banana green-ripening, the influence of high temperature on chlorophyll degradation at various ripening stages were investigated. After ripening initiation, the fruit were subjected to 8 different temperature changing regimes at both 20°C and 30°C in 5 days. The ripening and senescence parameters were monitored and relevant gene expression was analyzed by RNA gel blot hybridization. Temperature during the first 3 days, or precisely the stage around the on-set of the respiration peak, was found to be critical for color formation in bananas. When the fruit were held at 30°C at this stage, the fruit developed green-ripe with >30% of the original chlorophyll remaining, regardless the other stages were at 20°C. The repressed chlorophyll degradation by 30°C was positively correlated to the reduced expression of several key genes functioning in chlorophyll degradation, MaSGR, MaNYC and MaPaO, which behaved in a closely temperature responding manner. Conversely to chlorophyll degradation patterns, moving to 30°C induced fruit respiration rate, the expression of MaACO, an ethylene synthesis related gene, and accelerated the decrease in fruit firmness and chlorophyll florescence (Fv/Fm), indicating that the ripening of the fruit was accelerated at 30°C. Accordingly, in the context of fruit ripening but under not agreeable temperature (30°C), the expression of chlorophyll degradation genes in bananas was predominantly regulated by temperature, not responding to the fruit ripening proceeding. Interestingly, by using the alternating temperature system, we here the first time reported the sensitivity of chlorophyll degradation genes, MaSGR, MaPAO and MaNYC, to high temperature during banana fruit ripening.

A Major Role for the Plastid-Encoded RNA Polymerase Complex in the Expression of Plastid Transfer RNAs

Chloroplast transcription in land plants relies on collaboration between a plastid-encoded RNA polymerase (PEP) of cyanobacterial ancestry and a nucleus-encoded RNA polymerase of phage ancestry. PEP associates with additional proteins that are unrelated to bacterial transcription factors, many of which have been shown to be important for PEP activity in Arabidopsis (Arabidopsis thaliana). However, the biochemical roles of these PEP-associated proteins are not known. We describe phenotypes conditioned by transposon insertions in genes encoding the maize (Zea mays) orthologs of five such proteins: ZmPTAC2, ZmMurE, ZmPTAC10, ZmPTAC12, and ZmPRIN2. These mutants have similar ivory/virescent pigmentation and similar reductions in plastid ribosomes and photosynthetic complexes. RNA gel-blot and microarray hybridizations revealed numerous changes in plastid transcript populations, many of which resemble those reported for the orthologous mutants in Arabidopsis. However, unanticipated reductions in the abundance of numerous transfer RNAs (tRNAs) dominated the microarray data and were validated on RNA gel blots. The magnitude of the deficiencies for several tRNAs was similar to that of the most severely affected messenger RNAs, with the loss of trnL-UAA being particularly severe. These findings suggest that PEP and its associated proteins are critical for the robust transcription of numerous plastid tRNAs and that this function is essential for the prodigious translation of plastid-encoded proteins that is required during the installation of the photosynthetic apparatus.

Identification and Characterization of miRNA Transcriptome in Potato by High-Throughput Sequencing

Micro RNAs (miRNAs) represent a class of short, non-coding, endogenous RNAs which play important roles in post-transcriptional regulation of gene expression. While the diverse functions of miRNAs in model plants have been well studied, the impact of miRNAs in crop plant biology is poorly understood. Here we used high-throughput sequencing and bioinformatics analysis to analyze miRNAs in the tuber bearing crop potato (Solanum tuberosum). Small RNAs were analysed from leaf and stolon tissues. 28 conserved miRNA families were found and potato-specific miRNAs were identified and validated by RNA gel blot hybridization. The size, origin and predicted targets of conserved and potato specific miRNAs are described. The large number of miRNAs and complex population of small RNAs in potato suggest important roles for these non-coding RNAs in diverse physiological and metabolic pathways.

Small RNAs of the Bradyrhizobium/Rhodopseudomonas lineage and their analysis

Small RNAs (sRNAs) play a pivotal role in bacterial gene regulation. However, the sRNAs of the vast majority of bacteria with sequenced genomes still remain unknown since sRNA genes are usually difficult to recognize and thus not annotated. Here, expression of seven sRNAs (BjrC2a, BjrC2b, BjrC2c, BjrC68, BjrC80, BjrC174 and BjrC1505) predicted by genome comparison of Bradyrhizobium and Rhodopseudomonas members, was verified by RNA gel blot hybridization, microarray and deep sequencing analyses of RNA from the soybean symbiont Bradyrhizobium japonicum USDA 110. BjrC2a, BjrC2b and BjrC2c belong to the RNA family RF00519, while the other sRNAs are novel. For some of the sRNAs we observed expression differences between free-living bacteria and bacteroids in root nodules. The amount of BjrC1505 was decreased in nodules. By contrast, the amount of BjrC2a, BjrC68, BjrC80, BjrC174 and the previously described 6S RNA was increased in nodules, and accumulation of truncated forms of these sRNAs was observed. Comparative genomics and deep sequencing suggest that BjrC2a is an antisense RNA regulating the expression of inositol-monophosphatase. The analyzed sRNAs show a different degree of conservation in Rhizobiales, and expression of homologs of BjrC2, BjrC68, BjrC1505, and 6S RNA was confirmed in the free-living purple bacterium Rhodopseudomonas palustris 5D.

Identification and characterization of differentially expressed genes from tobacco roots after decapitation

Nicotine is the major alkaloid in tobacco and its biosynthesis is regulated by a variety of factors. Topping, as an important agronomic factor, can induce the nicotine biosynthesis increase. Some key nicotine biosynthesis-related genes and the framework of nicotine biosynthesis pathway have been well studied, but the details of nicotine biosynthesis pathway are not well understood now. To investigate the genes expressed after tobacco topping, we constructed a suppression subtractive hybridization library using cDNA from control tobacco plants as driver and those from topped tobacco plants as tester. The insert size of positive clones was 200–1,000 bp confirmed by PCR. After differential screening, 560 significantly differently expressed clones among 1,950 positive clones were acquired, sequenced and 273 high quality expressed sequence tags (ESTs) were acquired. The results of nucleotide blast homological analysis indicated that these ESTs mainly involved in alkaloid biosynthesis (4%), plant hormone metabolism (3%), signaling/transcription (18%), stress/defense (32%), protein metabolism (9%), carbon metabolism (6%), other metabolism (15%) and function unknown (13%). The expression of selected genes was analyzed by reverse transcription polymerase chain reaction and RNA gel blot hybridization, and the result indicated that their transcription amount increased after tobacco topping. NtNAC-R1was in silico cloned, and the expression level of NtNAC-R1 increased at 12 and 24 h in tobacco roots after topping, which indicated that NtNAC-R1 may play an important role in the signal transduction after tobacco topping. In addition to many previously reported nicotine biosynthesis-related genes, some new genes, such as transcription factors related to nicotine biosynthesis/regulation and the members of plant hormone pathway, were discovered in our library. The results contribute new data to the list of possible candidate genes involved in nicotine biosynthesis and regulation.

Functional Analysis of an Arabidopsis thaliana Abiotic Stress-inducible Facilitated Diffusion Transporter for Monosaccharides

Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus

The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22- or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.

Expression of rice Ca 2+-dependent protein kinases (CDPKs) genes under different environmental stresses

Ca 2+-dependent protein kinases (CDPKs) play an essential role in plant Ca 2+-mediated signal transduction. Twenty-nine CDPK genes have been identified in the rice genome through a complete search of genome and full-length cDNA databases. Eight of them were reported previously to be inducible by different stress stimuli. Sequence comparison revealed that all 29 CDPK genes ( OsCPK1-29) contain multiple stress-responsive cis-elements in the promoter region (1 kb) upstream of genes. Analysis of the information extracted from the Rice Expression Database indicates that 11 of the CDPK genes are regulated by chilling temperature, dehydration, salt, rice blast infection and chitin treatment. RT-PCR and RNA gel blot hybridization were performed in this study to detect the expression 19 of the CDPK genes. Twelve CDPK genes exhibited cultivar- and tissue-specific expression; four CDPK genes ( OsCPK6, OsCPK13, OsCPK17 and OsCPK25) were induced by chilling temperature, dehydration and salt stresses in the rice seedlings. While OsCPK13 ( OsCDPK7) was already known to be inducible by chilling temperature and high salt, this is the first report that the other three genes are stress-regulated. OsCPK6 and OsCPK25 are up-regulated by dehydration and heat shock, respectively, while OsCPK17 is down-regulated by chilling temperature, dehydration and high salt stresses. Based on this evidence, rice CDPK genes may be important components in the signal transduction pathways for stress responses. Findings from this research are important for further dissecting mechanisms of stress response and functions of CDPK genes in rice.

A novel gene, screened by cDNA-AFLP approach, contributes to lowering the acidity of fruit in apple

cDNA-AFLP, coupled with bulked segregant analysis (BSA), was used to screen genes expressed differently between low- and high-acid apple fruits from hybrids of ‘Toko’ × ‘Fuji’ ( Malus × domestica Borkh.). Sixty-four combinations of AFLP primers produced 2240 fragments, of which only one showed different expression between low- and high-acid fruits. The specific fragment was cloned and sequenced, and the complete cDNA was achieved by 3′ and 5′ rapid amplification of cDNA ends (RACE). The screened gene, designated as Mal-DDNA (GenBank accession no. DQ417661), showed no significant homology to clones in GenBank. The relatedness between fruit acidity and the transcription level of Mal-DDNA was identified by RT-PCR analysis on 30 hybrids. RT-PCR analysis indicated that Mal-DDNA transcripted in low-acid fruits at both early and ripe stages whereas in high- and mid-acid fruits, it did not transcript at the early stage. RNA gel-blot hybridization indicated that Mal-DDNA transcripted only in fruits and had clear difference between low- and high/mid-acid fruits. There was a good indication that Mal-DDNA existed as one copy in apple genome by Southern blot. Possible regulation of Mal-DDNA in apple fruit acidity is also discussed in the paper.

Dual Temporal Role of Plastid Sigma Factor 6 in Arabidopsis Development

Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription. The in vivo function of individual members of the sigma gene family has become increasingly accessible by knockout and complementation strategies. Here we have investigated plastid gene expression in an Arabidopsis (Arabidopsis thaliana) mutant with a defective gene for sigma factor 6. RNA gel-blot hybridization and real-time reverse transcription polymerase chain reaction together indicate that this factor has a dual developmental role, with both early and persistent (long-term) activities. The early role is evident from the sharp decrease of certain plastid transcripts only in young mutant seedlings. The second (persistent) role is reflected by the up- and down-regulation of other transcripts at the time of primary leaf formation and subsequent vegetative development. We conclude that sigma 6 does not represent a general factor, but seems to have specialized roles in developmental stage- and gene-specific plastid transcription. The possibility that plastid DNA copy number might be responsible for the altered transcript patterns in mutant versus wild type was excluded by the results of DNA gel-blot hybridization. Retransformation of the knockout line with the full-length sigma 6 cDNA further established a causal relationship between the functional sigma gene and the resulting phenotype.

Postharvest heat and conditioning treatments activate different molecular responses and reduce chilling injuries in grapefruit

A combination of hot water (a rinse at 62 degrees C for 20 s) and conditioning (pre-storage at 16 degrees C for 7 d) treatments synergistically reduced chilling injury development in grapefruit (Citrus paradisi, cv. "Star Ruby") during cold storage at 2 degrees C, suggesting that the treatments may activate different chilling tolerance responses. To study the molecular mechanisms involved, chilling- and conditioning-responsive genes were isolated by polymerase chain reaction (PCR) cDNA subtraction, cDNA libraries were constructed from hot water- and conditioning-treated fruit, and cDNA sequencing was used to identify putative stress-responsive and chilling tolerance genes. PCR cDNA subtraction revealed the identification of 17 chilling-responsive and heat- and conditioning-induced genes, and the expression patterns of 11 additional stress-related genes, antioxidant defensive genes, and genes encoding enzymes involved in membrane lipid modifications were characterized. It was found that hot water and conditioning treatments had little effect on gene expression by themselves, but rather had a priming effect, and enabled the fruit to activate their defence responses after subsequent exposure to chilling. RNA gel blot hybridizations revealed that the expression patterns of eight genes, including HSP19-I, HSP19-II, dehydrin, universal stress protein (USP), EIN2, 1,3;4-beta-D-glucanase, and superoxide dismutase (SOD), were specifically regulated by the heat treatment, and four genes, including fatty acid desaturase2 (FAD2) and lipid transfer protein (LTP), were specifically regulated by the conditioning treatment. Furthermore, four more genes were identified, including a translation initiation factor (SUI1), a chaperonin, and alcohol dehydrogenase (ADH), that were commonly regulated by both heat and conditioning treatments. According to these data, it is suggested that pre-storage heat and conditioning treatments may enhance fruit chilling tolerance by activating different molecular mechanisms. The hot water treatment activates mainly the expression of various stress-related genes, whereas the conditioning treatment activates mainly the expression of lipid membrane modification enzymes.

Characterization of a wheat histidine-containing phosphotransfer protein (HP) that is regulated by cytokinin-mediated inhibition of leaf senescence

A wheat histidine-containing phosphotransfer (HP) cDNA ( TaHP1) was isolated from a wheat seedling cDNA library using a RT-PCR product as a probe. The predicted amino acid sequence of TaHP1 is homologous to the histidine-containing phosphotransfer module of the multistep His-Asp phosphorelay from maize and Arabidopsis. The occurrence of HP in wheat was extensively inspected, two homologues were retrieved from wheat EST database and were named as TaHP2 and TaHP3, respectively. TaHP1, 2 and 3 are high homology and form as TaHP1 gene family. Phylogenetic analysis suggests that two classes of HP existed in plants. The secondary and three-dimensional structure analysis by molecular modeling revealed that the basic structure of plant HP proteins is similar with that of YDP1, a yeast HP protein. However, a slight difference in helix arrangement was found between TaHP1 and AHP1, the representative of class I and class II HP proteins, respectively. TaHP1 is present as a single copy gene in the wheat genome as demonstrated by DNA gel blot analysis. RNA gel blot hybridization demonstrated that TaHP1 family was actively expressed in seedlings, but could not be detected in root tissues. Treatment of leaf segments with BA induced TaHP1 family transcript accumulation in a dose-dependent manner, while treatment with trans-zeatin did not. These effects paralleled BA-mediated retardation leaf senescence. These results suggest that HP, like response regulator (RR), is also involved in the resetting or fine-tuning of cytokinin signal transduction and participation in cytokinin-mediated signaling during specific physiological processes, such as leaf senescence.

Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of l-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type-specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.

Genome‐wide identification of female‐enriched genes in zebrafish

Characteristic differences in morphology, physiology, and behavior between a male and female are correlated to the differential selection of sex-dependent transcriptomes. By using a cDNA array carrying approximately 9,000 zebrafish unique genes, we identified a group of genes whose expression are enriched in the female fish. A subset of these genes have been confirmed and further grouped as egg-enriched genes, as both somatic- and egg-enriched genes or as somatic-enriched genes by means of RNA gel blot hybridization. Most importantly, a significant proportion of these genes are either functionally unknown or are novel genes. Thus, future studies of this group of genes will help us greatly to understand more about sex-determination and sex-related physiology and behavior. In addition, comparison of zebrafish female-enriched genes with that in Drosophila, we found that only germline genes are shared between vertebrate and invertebrate, suggesting that the process of oogenesis is highly conserved during the evolution.