Autophagy targets Hd1 for vacuolar degradation to regulate rice flowering.

Flowering time (heading date) is a critical agronomic trait that determines the yield and regional adaptability of crops. Heading date 1 (Hd1) is a central regulator of photoperiodic flowering in rice (Oryza sativa). However, how the homeostasis of Hd1 protein is achieved is poorly understood. Here, we report that the nuclear autophagy pathway mediates Hd1 degradation in the dark to regulate flowering. Loss of autophagy function results in an accumulation of Hd1 and delays flowering under both short-day and long-day conditions. In the dark, nucleus-localized Hd1 is recognized as a substrate for autophagy and is subjected to vacuolar degradation via the autophagy protein OsATG8. The Hd1-OsATG8 interaction is required for autophagic degradation of Hd1 in the dark. Our study reveals a new mechanism by which Hd1 protein homeostasis is regulated by autophagy to control rice flowering. Our study also indicates that the regulation of flowering by autophagic degradation of Hd1 orthologs may have arisen over the course of mesangiosperm evolution, which would have increased their flexibility and adaptability to the environment by modulating flowering time.

Plastid Deficient 1 Is Essential for the Accumulation of Plastid-Encoded RNA Polymerase Core Subunit ß and Chloroplast Development in Arabidopsis.

Plastid-encoded RNA polymerase (PEP)-dependent transcription is an essential process for chloroplast development and plant growth. It is a complex event that is regulated by numerous nuclear-encoded proteins. In order to elucidate the complex regulation mechanism of PEP activity, identification and characterization of PEP activity regulation factors are needed. Here, we characterize Plastid Deficient 1 (PD1) as a novel regulator for PEP-dependent gene expression and chloroplast development in Arabidopsis. The PD1 gene encodes a protein that is conserved in photoautotrophic organisms. The Arabidopsis pd1 mutant showed albino and seedling-lethal phenotypes. The plastid development in the pd1 mutant was arrested. The PD1 protein localized in the chloroplasts, and it colocalized with nucleoid protein TRXz. RT-quantitative real-time PCR, northern blot, and run-on analyses indicated that the PEP-dependent transcription in the pd1 mutant was dramatically impaired, whereas the nuclear-encoded RNA polymerase-dependent transcription was up-regulated. The yeast two-hybrid assays and coimmunoprecipitation experiments showed that the PD1 protein interacts with PEP core subunit ß (PEP-ß), which has been verified to be essential for chloroplast development. The immunoblot analysis indicated that the accumulation of PEP-ß was barely detected in the pd1 mutant, whereas the accumulation of the other essential components of the PEP complex, such as core subunits α and ß', were not affected in the pd1 mutant. These observations suggested that the PD1 protein is essential for the accumulation of PEP-ß and chloroplast development in Arabidopsis, potentially by direct interaction with PEP-ß.

Developmental validation of the Microreader™ Y Prime Plus ID System: An advanced Y-STR 38-plex system for forensic applications.

Y-STR is widely used in sexual assaults and familial searches of suspects. Here, we reported a novel 38-plex STR genotyping system designed for forensic applications. Microreader™ Y Prime Plus ID System (YPP) amplifies 38 loci in one reaction, including 29 loci from commonly used Yfiler® Plus PCR Amplification Kit & PowerPlex® Y23 System (DYS393, DYS570, DYS19, DYS392, DYS549, Y GATA H4, DYS460, DYS458, DYS481, DYS635, DYS448, DYS533, DYS449, DYS456, DYS389I, DYS390, DYS389Ⅱ, DYS438, DYS391, DYS439, DYS437, DYS385a/b, DYS643, DYS518, DYS576, DYF387S1a/b, and DYS627), 6 commonly used loci for the Y-STR database (DYS444, DYS447, DYS596, DYF404a/b, DYS527a/b, DYS557) and one Y-indel specific for the Chinese population. YPP is designed for different types of samples, such as blood card and swabs. In this work, YPP was validated following SWGDAM guidelines (2016) and guidelines from Ministry of Public Security of the People's Republic of China, including PCR-based, sensitivity, accuracy and precision, mixture, stability and inhibitor, and species specificity. The results indicate that the Microreader™ Y Prime Plus ID System is a powerful identification kit designed for forensic databases.

Analysis of the changes of electron transfer and heterogeneity of photosystem II in Deg1-reduced Arabidopsis plants.

Deg1 protease functions in protease and chaperone of PSII complex components, but few works were performed to study the effects of Deg1 on electron transport activities on the donor and acceptor side of PSII and its correlation with the photoprotection of PSII during photoinhibition. Therefore, we performed systematic and comprehensive investigations of electron transfers on the donor and acceptor sides of photosystem II (PSII) in the Deg1-reduced transgenic lines deg1-2 and deg1-4. Both the maximal quantum efficiency of PSII photochemistry (Fv/Fm) and the actual PSII efficiency (ΦPSII) decreased significantly in the transgenic plants. Increases in nonphotochemical quenching (NPQ) and the dissipated energy flux per reaction center (DI0/RC) were also shown in the transgenic plants. Along with the decreased D1, CP47, and CP43 content, these results suggested photoinhibition under growth light conditions in transgenic plants. Decreased Deg1 caused inhibition of electron transfer on the PSII reducing side, leading to a decline in the number of QB-reducing centers and accumulation of QB-nonreducing centers. The Tm of the Q band shifted from 5.7 °C in the wild-type plant to 10.4 °C and 14.2 °C in the deg1-2 and deg1-4 plants, respectively, indicating an increase in the stability of S2QA¯ in transgenic plants. PSIIα in the transgenic plants largely reduced, while PSIIß and PSIIγ increased with the decline in the Deg1 levels in transgenic plants suggesting PSIIα centers gradually converted into PSIIß and PSIIγ centers in the transgenic plants. Besides, the connectivity of PSIIα and PSIIß was downregulated in transgenic plants. Our results reveal that downregulation of Deg1 protein levels induced photoinhibition in transgenic plants, leading to loss of PSII activities on both the donor and acceptor sides in transgenic plants. These results give a new insight into the regulation role of Deg1 in PSII electron transport.

OsSWEET14 cooperates with OsSWEET11 to contribute to grain filling in rice.

The grain-filling process is crucial for cereal crop yields, but how the caryopsis of such plants is supplied with sugars, which are produced by photosynthesis in leaves and then transported long distance, is largely unknown. In rice (Oryza sativa), various SWEET family sucrose transporters are thought to have important roles in grain filling. Here, we report that OsSWEET14 plays a crucial part in this process in rice. ossweet14 knockout mutants did not show any detectable phenotypic differences from the wild type, whereas ossweet14;ossweet11 double-knockout mutants had much more severe phenotypes than ossweet11 single-knockout mutants, including strongly reduced grain weight and yield, reduced grain-filling rate, and increased starch accumulation in the pericarp. Both OsSWEET14 and OsSWEET11 exhibited distinct spatiotemporal expression patterns between the early stage of caryopsis development and the rapid grain-filling stage. During the rapid grain-filling stage, OsSWEET14 and OsSWEET11 localized to four key sites: vascular parenchyma cells, the nucellar projection, the nucellar epidermis, and cross cells. These results demonstrate that OsSWEET14 plays an important role in grain filling, and they suggest that four major apoplasmic pathways supply sucrose to the endosperm during the rapid grain-filling stage via the sucrose effluxers SWEET14 and SWEET11.

Pentatricopeptide repeat protein PHOTOSYSTEM I BIOGENESIS FACTOR2 is required for splicing of ycf3.

To gain a better understanding of the molecular mechanisms of photosystem I (PSI) biogenesis, we characterized the Arabidopsis thaliana photosystem I biogenesis factor 2 (pbf2) mutant, which lacks PSI complex. PBF2 encodes a P-class pentatricopeptide repeat (PPR) protein. In the pbf2 mutants, we observed a striking decrease in the transcript level of only one gene, the chloroplast gene ycf3, which is essential for PSI assembly. Further analysis of ycf3 transcripts showed that PBF2 is specifically required for the splicing of ycf3 intron 1. Computational prediction of binding sequences and electrophoretic mobility shift assays reveal that PBF2 specifically binds to a sequence in ycf3 intron 1. Moreover, we found that PBF2 interacted with two general factors for group II intron splicing CHLOROPLAST RNA SPLICING2-ASSOCIATED FACTOR1 (CAF1) and CAF2, and facilitated the association of these two factors with ycf3 intron 1. Our results suggest that PBF2 is specifically required for the splicing of ycf3 intron 1 through cooperating with CAF1 and CAF2. Our results also suggest that additional proteins are required to contribute to the specificity of CAF-dependent group II intron splicing.

Validation of the Microreader™ 29Y Prime ID system for forensic use.

Currently, Y-short tandem repeat loci (Y-STRs) have been increasingly used in the forensic field, particularly in investigations of sexual assault, determination of paternity and male lineage studies because of the characteristics of male-only and paternal inheritance. The Microreader™ 29Y Prime ID system is a 29-plex Y-STR genotyping system that amplifies 17 widely used commercial loci (DYS570, DYS546, DYS460, DYS458, DYS635, DYS533, DYS448, DYS627, DYS456, DYS576, DYS449, DYS437, DYS643, DYS518, DYF387S1 a/b, and a sexual locus Y GATA H4), European recommended 7 single-copy "minimal haplotypes" (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, and DYS385a/b) and 2 additional loci (DYS438 and DYS439) recommended by The Scientific Working Group on DNA Analysis Methods (SWGDAM). The Microreader™ 29Y Prime ID system was validated according to the guidelines of "Validation Guidelines for DNA Analysis Methods (2016)" described by the Scientific Working Group on DNA Analysis Methods (SWGDAM), including PCR-based, sensitivity, precision and accuracy evaluation, stutter percentage and peak height ratio, inhibitors, species specificity and DNA mixture studies. This study indicates that the Microreader™ 29Y Prime ID system is a useful tool for forensic cases and Y-STR genotyping.

Storage quality of walnut oil containing lycopene during accelerated oxidation.

The purpose of investigation was to assess the effect of lycopene on the peroxide value, acid value, fatty acids, total phenolic content and ferric-reducing antioxidant power of walnut oil. Walnut oil was extracted from Xinjiang walnut variety using cold pressing method. Our study reported that after 45 days of accelerated oxidation at 60 °C (Schaal oven test), 0.005% lycopene exhibited the greatest antioxidant effect than other addition levels of lycopene. Therefore, under ambient storage conditions, the shelf-life of walnut oil could be extended up to 16 months by 0.005% lycopene. Moreover, 0.005% lycopene added to walnut oil had a significantly higher content of saturated fatty acid, unsaturated fatty acid, total phenol, reducing ability of the polar and non-polar components than the blank sample (walnut oil without any addition of lycopene). In conclusion, lycopene improved the quality of walnut oil because of its antioxidant effect against lipid oxidation.

The Phytol Phosphorylation Pathway Is Essential for the Biosynthesis of Phylloquinone, which Is Required for Photosystem I Stability in Arabidopsis.

Phytyl-diphosphate, which provides phytyl moieties as a common substrate in both tocopherol and phylloquinone biosynthesis, derives from de novo isoprenoid biosynthesis or a salvage pathway via phytol phosphorylation. However, very little is known about the role and origin of the phytyl moiety for phylloquinone biosynthesis. Since VTE6, a phytyl-phosphate kinase, is a key enzyme for phytol phosphorylation, we characterized Arabidopsis vte6 mutants to gain insight into the roles of phytyl moieties in phylloquinone biosynthesis and of phylloquinone in photosystem I (PSI) biogenesis. The VTE6 knockout mutants vte6-1 and vte6-2 lacked detectable phylloquinone, whereas the phylloquinone content in the VTE6 knockdown mutant vte6-3 was 90% lower than that in wild-type. In vte6 mutants, PSI function was impaired and accumulation of the PSI complex was defective. The PSI core subunits PsaA/B were efficiently synthesized and assembled into the PSI complex in vte6-3. However, the degradation rate of PSI subunits in the assembled PSI complex was more rapid in vte6-3 than in wild-type. In vte6-3, PSI was more susceptible to high-light damage than in wild-type. Our results provide the first genetic evidence that the phytol phosphorylation pathway is essential for phylloquinone biosynthesis, and that phylloquinone is required for PSI complex stability.

Decreased glutathione reductase2 leads to early leaf senescence in Arabidopsis.

Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) and participates in the ascorbate-glutathione cycle, which scavenges H2 O2 . Here, we report that chloroplastic/mitochondrial GR2 is an important regulator of leaf senescence. Seed development of the homozygous gr2 knockout mutant was blocked at the globular stage. Therefore, to investigate the function of GR2 in leaf senescence, we generated transgenic Arabidopsis plants with decreased GR2 using RNAi. The GR2 RNAi plants displayed early onset of age-dependent and dark- and H2 O2 -induced leaf senescence, which was accompanied by the induction of the senescence-related marker genes SAG12 and SAG13. Furthermore, transcriptome analysis revealed that genes related to leaf senescence, oxidative stress, and phytohormone pathways were upregulated directly before senescence in RNAi plants. In addition, H2 O2 accumulated to higher levels in RNAi plants than in wild-type plants and the levels of H2 O2 peaked in RNAi plants directly before the early onset of leaf senescence. RNAi plants showed a greater decrease in GSH/GSSG levels than wild-type plants during leaf development. Our results suggest that GR2 plays an important role in leaf senescence by modulating H2 O2 and glutathione signaling in Arabidopsis.

Purine biosynthetic enzyme ATase2 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis.

To investigate the molecular mechanism of chloroplast biogenesis and development, we characterized an Arabidopsis mutant (dg169, delayed greening 169) which showed growth retardation and delayed greening phenotype in leaves. Newly emerged chlorotic leaves recovered gradually with leaf development in the mutant, and the mature leaves showed similar phenotype to those of wild-typewild-type plants. Compared with wild-type, the chloroplasts were oval-shaped and smaller and the thylakoid membranes were less abundant in yellow section of young leaves of dg169. In addition, the functions of photosystem II (PSII) and photosystem I (PSI) were also impaired. Furthermore, the amount of core subunits of PSII and PSI, as well as PSII and PSI complexes reduced in yellow section of young leaves of dg169. Map-based positional cloning identified that phenotype of dg169 was attributed to a point mutation of ATase2 which converts the conserved Ile-155 residue to Asn. ATase2 catalyzes the first step of de novo purine biosynthesis. This mutation resulted in impaired purine synthesis and a significant decrease in ATP, ADP, GTP and GDP contents. The analysis of ATase2-GFP protein fusion showed that ATase2 was localized to nucleoid of chloroplasts. Our results further demonstrated that the levels of PEP-dependent transcripts in yellow section of young leaves of dg169 were decreased while NEP-dependent and both PEP- and NEP-dependent transcripts and chloroplast DNA replications were increased. The results in this study suggest that ATase2 plays an essential role in early chloroplast development through maintaining PEP function.

Functional analysis of the rice rubisco activase promoter in transgenic Arabidopsis.

To gain a better understanding of the regulatory mechanism of the rice rubisco activase (Rca) gene, variants of the Rca gene promoter (one full-length and four deletion mutants) fused to the coding region of the bacterial reporter gene ß-glucuronidase (GUS) were introduced into Arabidopsis via Agrobacterium-mediated transformation. Our results show that a 340 bp fragment spanning from -297 to +43 bp relative to the transcription initiation site is enough to promote tissue-specific and light-inducible expression of the rice Rca gene as done by the full-length promoter (-1428 to +43 bp). Further deletion analysis indicated that the region conferring tissue-specificity of Rca expression is localized within a 105 bp fragment from -58 to +43 bp, while light-inducible expression of Rca is mediated by the region from -297 to -58 bp. Gel shift assays and competition experiments demonstrated that rice nuclear proteins bind specifically with the fragment conferring light responsiveness at more than one binding site. This implies that multiple cis-elements may be involved in light-induced expression of the rice Rca gene. These works provide a useful reference for understanding transcriptional regulation mechanism of the rice Rca gene, and lay a strong foundation for further detection of related cis-elements and trans-factors.

Characterization of photosystem II in transgenic tobacco plants with decreased iron superoxide dismutase.

Iron superoxide dismutases (FeSODs) play an important role in preventing the oxidative damage associated with photosynthesis. To investigate the mechanisms of FeSOD in protection against photooxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with severely decreased FeSOD by using a gene encoding tobacco chloroplastic FeSOD for the RNAi construct. Transgenic plants were highly sensitive to photooxidative stress and accumulated increased levels of O2•⁻ under normal light conditions. Spectroscopic analysis and electron transport measurements showed that PSII activity was significantly reduced in transgenic plants. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed that there was a slow electron transfer between Q(A) and Q(B) and decreased redox potential of Q(B) in transgenic plants, whereas the donor side function of PSII was not affected. Immunoblot and blue native gel analyses showed that PSII protein accumulation was also decreased in transgenic plants. PSII photodamage and D1 protein degradation under high light treatment was increased in transgenic plants, whereas the PSII repair was not affected, indicating that the stability of the PSII complex was decreased in transgenic plants. The results in this study suggest that FeSOD plays an important role in maintaining PSII function by stabilizing PSII complexes in tobacco plants.

Characterization of photosystem II photochemistry in transgenic tobacco plants with lowered Rubisco activase content.

Rubisco activase plays an important role in the regulation of CO(2) assimilation. However, it is unknown how activase regulates photosystem II (PSII) photochemistry. To investigate the effects of Rubisco activase on PSII photochemistry, we obtained transgenic tobacco (Nicotiana tabacum) plants with 50% (i7), 25% (i28), and 5% (i46) activase levels as compared to wild type plants by using a gene encoding tobacco activase for the RNAi construct. Both CO(2) assimilation and PSII activity were significantly reduced only in transgenic i28 and i46 plants, suggesting that activase deficiency led to decreased PSII activity. Flash-induced fluorescence kinetics indicated that activase deficiency resulted in a slow electron transfer between Q(A) (primary quinine electron acceptor of PSII) and Q(B) (secondary quinone electron acceptor of PSII). Thermoluminescence measurements revealed that activase deficiency induced a shift of S(2)Q(A)(-) and S(2)Q(B)(-) recombinations to higher temperatures in parallel, and a decrease in the intensities of the thermoluminescence emissions. Activase deficiency also dampened the period-four oscillation of the thermoluminescence B-band. Protein gel blot analysis showed that activase deficiency resulted in a significant decrease in the content of D1, D2, CP43, CP47, and PsbO proteins. Transmission electron microscopy analysis demonstrated that activase deficiency induced a significant decrease in the number of grana stacks per chloroplast and discs per grana stack. Our results suggest that activase plays an important role in maintaining PSII function and chloroplast development.

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state.

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30-70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO(2) assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H(2)O(2) in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.

Genetic engineering of the biosynthesis of glycinebetaine enhances thermotolerance of photosystem II in tobacco plants.

Genetically engineered tobacco (Nicotiana tabacum L.) with the ability to accumulate glycinebetaine was established. The wild type and transgenic plants were exposed to heat treatment (25-50 degrees C) for 4 h in the dark and under growth light intensity (300 mumol m(-2) s(-1)). The analyses of oxygen-evolving activity and chlorophyll fluorescence demonstrated that photosystem II (PSII) in transgenic plants showed higher thermotolerance than in wild type plants in particular when heat stress was performed in the light, suggesting that the accumulation of glycinebetaine leads to increased tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement on thermostability of the oxygen-evolving complex and the reaction center of PSII. The enhanced tolerance was caused by acceleration of the repair of PSII from heat-enhanced photoinhibition. Under heat stress, there was a significant accumulation of H(2)O(2), O (2) (-) and catalytic Fe in wild type plants but this accumulation was much less in transgenic plants. Heat stress significantly decreased the activities of catalase, ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase in wild type plants whereas the activities of these enzymes either decreased much less or maintained or even increased in transgenic plants. In addition, heat stress increased the activity of superoxide dismutase in wild type plants but this increase was much greater in transgenic plants. Furthermore, transgenic plants also showed higher content of ascorbate and reduced glutathione than that of wild type plants under heat stress. The results suggest that the increased thermotolerance induced by accumulation of glycinebetaine in vivo was associated with the enhancement of the repair of PSII from heat-enhanced photo inhibition, which might be due to less accumulation of reactive oxygen species in transgenic plants.

Heat stress induces an aggregation of the light-harvesting complex of photosystem II in spinach plants.

Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25 degrees C-50 degrees C) in the dark for 30 min. At temperatures higher than 35 degrees C, CO2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45 degrees C and decreased only slightly at 50 degrees C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25 degrees C to 50 degrees C in the dark for 10 min. At temperatures over 35 degrees C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25 degrees C to 50 degrees C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35 degrees C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.

Sucrose regulates elongation of carrot somatic embryo radicles as a signal molecule.

Elongation of carrot somatic embryo radicles was inhibited by sucrose at or above 5% (145 mM). This effect would not be released until the sucrose concentration was lowered again. Morphological and cytological studies as well as determination of ABA content and analysis of the expression mode of a Lea gene, all point to its similarity to natural dormancy and germination of seeds. Use of monosaccharides (glucose and fructose), other disaccharide (maltose), and isomolar concentration of osmotica (mannitol and sorbitol), did not show similar regulatory effect. It is thus clear that the regulatory effect is not a result of simple osmotic stress. Hexokinase inhibitors such as glucosamine and N -acetyl-glucosamine did not exert any influence on the regulation-deregulation effects of sucrose. Mannose, which inhibits germination of Arabidopsis seeds, did not prevent carrot somatic embryo radicles from elongating. It is thus inferred that this sucrose-signaling pathway may be independent of hexokinase. As a first step to understand the molecular mechanism of this process, a carrot sucrose transporter gene ( cSUT ) expressed in the embryos and roots specifically was isolated. Studies on transformed yeast mutant with cSUT cDNA identified its sucrose transport activity. Northern hybridization and gel retardation experiment revealed that there is a marked increase in expression of cSUT at the beginning of somatic embryo germination, and this is attributed to regulation on the level of transcription. This suggested the possibility that cSUT has an important role in this sucrose signal regulation system.

Molecular characterization of poly(A)-binding protein from Daucus carota.

Poly(A)-binding proteins (PABs) bind to the poly(A) tails of most eukaryotic mRNAs and thereby influence the translational efficiency as well as the stability of the mRNAs thus bound. Compared with the data on yeast PAB, relatively little is known about the functions of PABs in higher plants. The cDNA encoding PAB was cloned by the method of "virtual subtraction" from carrot somatic embryos cultured with different sucrose concentrations. Sequence alignment reveals a significant homology between the deduced amino acid sequence of DcPAB and those of other PABs. The deduced sequence consists of 658 amino acids with a calculated molecular weight of 71.9 kDa. The cDNA has been expressed as a recombinant protein in Escherichia coli. The differential expression of DcPAB under different conditions suggests a potently unique role in the development of carrot somatic embryo.