Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

From seed germination to flowering, light controls plant development via the pigment phytochrome.

Plant growth and development are regulated by interactions between the environment and endogenous developmental programs. Of the various environmental factors controlling plant development, light plays an especially important role, in photosynthesis, in seasonal and diurnal time sensing, and as a cue for altering developmental pattern. Recently, several laboratories have devised a variety of genetic screens using Arabidopsis thaliana to dissect the signal transduction pathways of the various photoreceptor systems. Genetic analysis demonstrates that light responses are not simply endpoints of linear signal transduction pathways but are the result of the integration of information from a variety of photoreceptors through a complex network of interacting signaling components. These signaling components include the red/far-red light receptors, phytochromes, at least one blue light receptor, and negative regulatory genes (DET, COP, and FUS) that act downstream from the photoreceptors in the nucleus. In addition, a steroid hormone, brassinolide, also plays a role in light-regulated development and gene expression in Arabidopsis. These molecular and genetic data are allowing us to construct models of the mechanisms by which light controls development and gene expression in Arabidopsis. In the future, this knowledge can be used as a framework for understanding how all land plants respond to changes in their environment.

Induction of HSP70 and polyubiquitin expression associated with plant virus replication

By examining the front of virus invasion in immature pea embryos infected with pea seed-borne mosaic virus (PSbMV), the selective control of different host genes has been observed. From our observations, the early responses to PSbMV replication can be grouped into three classes, inhibited host gene expression, induced host gene expression, and no effect on a normal host function. The expression of two heat-inducible genes encoding HSP70 and polyubiquitin was induced coordinately with the onset of virus replication and the down-regulation of two other genes encoding lipoxygenase and heat shock cognate protein. The down-regulation was part of a general suppression of host gene expression that may be achieved through the degradation of host transcripts. We discuss the possibilities of whether the induction of HSP70 and polyubiquitin genes represents a requirement for the respective protein products by the virus or is merely a consequence of the depletion of other host transcripts. The former is feasible, as the induction of both genes does result in increased HSP70 and ubiquitin accumulation. This also indicates that, in contrast to some animal virus infections, there is not a general inhibition of translation of host mRNAs following PSbMV infection. This selective control of host gene expression was observed in all cell types of the embryo and identifies mechanisms of cellular disruption that could act as triggers for symptom expression.

Directed seed dispersal by bellbirds in a tropical cloud forest

A fundamental goal of plant population ecology is to understand the consequences for plant fitness of seed dispersal by animals. Theories of seed dispersal and tropical forest regeneration suggest that the advantages of seed dispersal for most plants are escape from seed predation near the parent tree and colonization of vacant sites, the locations of which are unpredictable in space and time. Some plants may gain in fitness as a fortuitous consequence of disperser behavior if certain species of dispersers nonrandomly place seeds in sites predictably favorable for seedling establishment. Such patterns of directed dispersal by vertebrates long have been suggested but never demonstrated for tropical forest trees. Here we report the pattern of seed distribution and 1-year seedling survival generated by five species of birds for a neotropical, shade-tolerant tree. Four of the species dispersed seeds to sites near the parent trees with microhabitat characteristics similar to those at random locations, whereas the fifth species, a bellbird, predictably dispersed seeds under song perches in canopy gaps. The pattern of seedling recruitment was bimodal, with a peak near parent trees and a second peak, corresponding to bellbird song perches, far (>40 m) from parent trees. Seedling survival was higher for seeds dispersed by bellbirds than by the other species, because of a reduction in seedling mortality by fungal pathogens in gaps. Thus, bellbirds play a significant role in seed dispersal by providing directed dispersal to favorable sites and therefore may influence plant recruitment patterns and species diversity in Neotropical forests.

Mendel-GFDb and Mendel-ESTS: databases of plant gene families and ESTs annotated with gene family numbers and gene family names

There is no control over the information provided with sequences when they are deposited in the sequence databases. Consequently mistakes can seed the incorrect annotation of other sequences. Grouping genes into families and applying controlled annotation overcomes the problems of incorrect annotation associated with individual sequences. Two databases (http://www.mendel.ac.uk) were created to apply controlled annotation to plant genes and plant ESTs: Mendel-GFDb is a database of plant protein (gene) families based on gapped-BLAST analysis of all sequences in the SWISS-PROT family of databases. Sequences are aligned (ClustalW) and identical and similar residues shaded. The families are visually curated to ensure that one or more criteria, for example overall relatedness and/or domain similarity relate all sequences within a family. Sequence families are assigned a ‘Gene Family Number’ and a unified description is developed which best describes the family and its members. If authority exists the gene family is assigned a ‘Gene Family Name’. This information is placed in Mendel-GFDb. Mendel-ESTS is primarily a database of plant ESTs, which have been compared to Mendel-GFDb, completely sequenced genomes and domain databases. This approach associated ESTs with individual sequences and the controlled annotation of gene families and protein domains; the information being placed in Mendel-ESTS. The controlled annotation applied to genes and ESTs provides a basis from which a plant transcription database can be developed.

Structure of a flavin-binding plant photoreceptor domain: Insights into light-mediated signal transduction

Phototropin, a major blue-light receptor for phototropism in seed plants, exhibits blue-light-dependent autophosphorylation and contains two light, oxygen, or voltage (LOV) domains and a serine/threonine kinase domain. The LOV domains share homology with the PER-ARNT-SIM (PAS) superfamily, a diverse group of sensor proteins. Each LOV domain noncovalently binds a single FMN molecule and exhibits reversible photochemistry in vitro when expressed separately or in tandem. We have determined the crystal structure of the LOV2 domain from the phototropin segment of the chimeric fern photoreceptor phy3 to 2.7-Å resolution. The structure constitutes an FMN-binding fold that reveals how the flavin cofactor is embedded in the protein. The single LOV2 cysteine residue is located 4.2 Å from flavin atom C(4a), consistent with a model in which absorption of blue light induces formation of a covalent cysteinyl-C(4a) adduct. Residues that interact with FMN in the phototropin segment of the chimeric fern photoreceptor (phy3) LOV2 are conserved in LOV domains from phototropin of other plant species and from three proteins involved in the regulation of circadian rhythms in Arabidopsis and Neurospora. This conservation suggests that these domains exhibit the same overall fold and share a common mechanism for flavin binding and light-induced signaling.

Cloning and Characterization of TPE4A, a Thiol-Protease Gene Induced during Ovary Senescence and Seed Germination in Pea1

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

Temporal and Spatial Patterns of Accumulation of the Transcript of Myo-Inositol-1-Phosphate Synthase and Phytin-Containing Particles during Seed Development in Rice1

Myo-inositol-1-phosphate (I[1]P) synthase (EC 5.5.1.4) catalyzes the reaction from glucose 6-phosphate to I(1)P, the first step of myo-inositol biosynthesis. Among the metabolites of I(1)P is inositol hexakisphosphate, which forms a mixed salt called phytin or phytate, a storage form of phosphate and cations in seeds. We have isolated a rice (Oryza sativa L.) cDNA clone, pRINO1, that is highly homologous to the I(1)P synthase from yeast and plants. Northern analysis of total RNA showed that the transcript accumulated to high levels in embryos but was undetectable in shoots, roots, and flowers. In situ hybridization of developing seeds showed that the transcript first appeared in the apical region of globular-stage embryos 2 d after anthesis (DAA). Strong signals were detected in the scutellum and aleurone layer after 4 DAA. The level of the transcript in these cells increased until 7 DAA, after which time it gradually decreased. Phytin-containing particles called globoids appeared 4 DAA in the scutellum and aleurone layer, coinciding with the localization of the RINO1 transcript. The temporal and spatial patterns of accumulation of the RINO1 transcript and globoids suggest that I(1)P synthase directs phytin biosynthesis in rice seeds.

Age-Induced Protein Modifications and Increased Proteolysis in Potato Seed-Tubers1

Long-term aging of potato (Solanum tuberosum) seed-tubers resulted in a loss of patatin (40 kD) and a cysteine-proteinase inhibitor, potato multicystatin (PMC), as well as an increase in the activities of 84-, 95-, and 125-kD proteinases. Highly active, additional proteinases (75, 90, and 100 kD) appeared in the oldest tubers. Over 90% of the total proteolytic activity in aged tubers was sensitive to trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane or leupeptin, whereas pepstatin was the most effective inhibitor of proteinases in young tubers. Proteinases in aged tubers were also inhibited by crude extracts or purified PMC from young tubers, suggesting that the loss of PMC was responsible for the age-induced increase in proteinase activity. Nonenzymatic oxidation, glycation, and deamidation of proteins were enhanced by aging. Aged tubers developed “daughter” tubers that contained 3-fold more protein than “mother” tubers, with a polypeptide profile consistent with that of young tubers. Although PMC and patatin were absent from the older mother tubers, both proteins were expressed in the daughter tubers, indicating that aging did not compromise the efficacy of genes encoding PMC and patatin. Unlike the mother tubers, proteinase activity in daughter tubers was undetectable. Our results indicate that tuber aging nonenzymatically modifies proteins, which enhances their susceptibility to breakdown; we also identify a role for PMC in regulating protein turnover in potato tubers.

Plant genome values: How much do we know?

Plants are the basis of life on earth. We cannot overemphasize their importance. The value of plant genome initiatives is self-evident. The need is to identify priorities for action. The angiosperm genome is highly variable, but the extent of this variability is unknown. Uncertainties remain about the number of genes and the number of species living. Many plants will become extinct before they are discovered. We risk losing both genes and vital information about plant uses. There are also major gaps in our karyotypic knowledge. No chromosome count exists for >70% of angiosperm species. DNA C values are known for only ≈1% of angiosperms, a sample unrepresentative of the global flora. Researchers reported new relationships between genome size and characters of major interest for plant breeding and the environment and the need for more data. In 1997, a Royal Botanic Gardens Kew workshop identified gaps and planned international collaboration to fill them. An electronic version of the Angiosperm DNA C value database also was published. Another initiative, which will make a very significant contribution to the conservation of plant genetic diversity on a global scale is Kew’s Millennium Seed Bank, partly funded by the U.K. Millennium Commission, celebrating the year 2000. Costing up to £80 million (£1 = $1.62), its main aims are to collect and conserve the seed of almost all of the U.K. spermatophyte flora by the year 2000, to collect and conserve a further 10% of the world spermatophyte flora principally from the drylands by 2009, and to provide a world class building as the focus of this activity by 2000.

A Fiberless Seed Mutation in Cotton Is Associated with Lack of Fiber Cell Initiation in Ovule Epidermis and Alterations in Sucrose Synthase Expression and Carbon Partitioning in Developing Seeds1

Fiber cell initiation in the epidermal cells of cotton (Gossypium hirsutum L.) ovules represents a unique example of trichome development in higher plants. Little is known about the molecular and metabolic mechanisms controlling this process. Here we report a comparative analysis of a fiberless seed (fls) mutant (lacking fibers) and a normal (FLS) mutant to better understand the initial cytological events in fiber development and to analyze the metabolic changes that are associated with the loss of a major sink for sucrose during cellulose biosynthesis in the mutant seeds. On the day of anthesis (0 DAA), the mutant ovular epidermal cells lacked the typical bud-like projections that are seen in FLS ovules and are required for commitment to the fiber development pathway. Cell-specific gene expression analyses at 0 DAA showed that sucrose synthase (SuSy) RNA and protein were undetectable in fls ovules but were in abundant, steady-state levels in initiating fiber cells of the FLS ovules. Tissue-level analyses of developing seeds 15 to 35 DAA revealed an altered temporal pattern of SuSy expression in the mutant relative to the normal genotype. Whether the altered programming of SuSy expression is the cause or the result of the mutation is unknown. The developing seeds of the fls mutant have also shown several correlated changes that represent altered carbon partitioning in seed coats and cotyledons as compared with the FLS genotype.

wrinkled1: A Novel, Low-Seed-Oil Mutant of Arabidopsis with a Deficiency in the Seed-Specific Regulation of Carbohydrate Metabolism1

During oil deposition in developing seeds of Arabidopsis, photosynthate is imported in the form of carbohydrates into the embryo and converted to triacylglycerols. To identify genes essential for this process and to investigate the molecular basis for the developmental regulation of oil accumulation, mutants producing wrinkled, incompletely filled seeds were isolated. A novel mutant locus, wrinkled1 (wri1), which maps to the bottom of chromosome 3 and causes an 80% reduction in seed oil content, was identified. Wild-type and homozygous wri1 mutant plantlets or mature plants were indistinguishable. However, developing homozygous wri1 seeds were impaired in the incorporation of sucrose and glucose into triacylglycerols, but incorporated pyruvate and acetate at an increased rate. Because the activities of several glycolytic enzymes, in particular hexokinase and pyrophosphate-dependent phosphofructokinase, are reduced in developing homozygous wri1 seeds, it is suggested that WRI1 is involved in the developmental regulation of carbohydrate metabolism during seed filling.

Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.

A single mutation that increases maize seed weight.

The maize endosperm-specific gene shrunken2 (Sh2) encodes the large subunit of the heterotetrameric starch synthetic enzyme adenosine diphosphoglucose pyrophosphorylase (AGP; EC 2.7.7.27). Here we exploit an in vivo, site-specific mutagenesis system to create short insertion mutations in a region of the gene known to be involved in the allosteric regulation of AGP. The site-specific mutagen is the transposable element dissociation (Ds). Approximately one-third (8 of 23) of the germinal revertants sequenced restored the wild-type sequence, whereas the remaining revertants contained insertions of 3 or 6 bp. All revertants retained the original reading frame 3' to the insertion site and involved the addition of tyrosine and/or serine. Each insertion revertant reduced total AGP activity and the amount of the SH2 protein. The revertant containing additional tyrosine and serine residues increased seed weight 11-18% without increasing or decreasing the percentage of starch. Other insertion revertants lacking an additional serine reduced seed weight. Reduced sensitivity to phosphate, a long-known inhibitor of AGP, was found in the high seed-weight revertant. This alteration is likely universally important since insertion of tyrosine and serine in the potato large subunit of AGP at the comparable position and expression in Escherichia coli also led to a phosphate-insensitive enzyme. These results show that single gene mutations giving rise to increased seed weight, and therefore perhaps yield, are clearly possible in a plant with a long history of intensive and successful breeding efforts.