Tony Arioli

Toward Sequencing Cotton (Gossypium) Genomes

Despite rapidly decreasing costs and innovative technologies, sequencing of angiosperm genomes is not yet undertaken lightly. Generating larger amounts of sequence data more quickly does not address the difficulties of sequencing and assembling complex genomes de novo. The cotton ( Gossypium spp.)

Meta-analysis of cotton fiber quality QTLs across diverse environments in a Gossypium hirsutum x G. barbadense RIL population

BackgroundCotton fibers (produced by Gossypium species) are the premier natural fibers for textile production. The two tetraploid species, G. barbadense (Gb) and G. hirsutum (Gh), differ significantly in their fiber properties, the former having much longer, finer and stronger fibers that are highly prized. A better understanding of the genetics and underlying biological causes of these differences will aid further improvement of cotton quality through breeding and biotechnology. We evaluated an inter-specific Gh × Gb recombinant inbred line (RIL) population for fiber characteristics in 11 independent experiments under field and glasshouse conditions. Sites were located on 4 continents and 5 countries and some locations were analyzed over multiple years.ResultsThe RIL population displayed a large variability for all major fiber traits. QTL analyses were performed on a per-site basis by composite interval mapping. Among the 651 putative QTLs (LOD > 2), 167 had a LOD exceeding permutation based thresholds. Coincidence in QTL location across data sets was assessed for the fiber trait categories strength, elongation, length, length uniformity, fineness/maturity, and color. A meta-analysis of more than a thousand putative QTLs was conducted with MetaQTL software to integrate QTL data from the RIL and 3 backcross populations (from the same parents) and to compare them with the literature. Although the global level of congruence across experiments and populations was generally moderate, the QTL clustering was possible for 30 trait x chromosome combinations (5 traits in 19 different chromosomes) where an effective co-localization of unidirectional (similar sign of additivity) QTLs from at least 5 different data sets was observed. Most consistent meta-clusters were identified for fiber color on chromosomes c6, c8 and c25, fineness on c15, and fiber length on c3.ConclusionsMeta-analysis provided a reliable means of integrating phenotypic and genetic mapping data across multiple populations and environments for complex fiber traits. The consistent chromosomal regions contributing to fiber quality traits constitute good candidates for the further dissection of the genetic and genomic factors underlying important fiber characteristics, and for marker-assisted selection.

Transcript profiling during fiber development identifies pathways in secondary metabolism and cell wall structure that may contribute to cotton fiber quality.

A global gene expression profiling study at different stages of fiber development was undertaken on two cotton species cultivated for fiber, Gossypium hirsutum (L.) and G. barbadense (L.). A large proportion of the genome was expressed during both fiber elongation and subsequent secondary cell wall thickening. There was a major shift in abundance of transcripts for gene regulation, cell organization and metabolism between fiber elongation and fiber thickening that was fundamentally similar in both species. Each stage had its own distinctive features represented by specific metabolic and regulatory genes, a number of which have been noted previously. Many of the genes expressed in the fibers were of a similar type and developmental expression to those seen in other fiber-producing plants, indicating a conservation of mechanisms of cell elongation and wall thickening across diverse plant genera. Secondary metabolism and pectin synthesis and modification genes were amongst the most statistically significant differentially expressed categories between the two species during fiber elongation. The gene profiles of the fiber thickening stage, however, were almost identical between the two species, suggesting that their different final fiber quality properties may be established at earlier stages of fiber development. Expression levels of representative phenylpropanoid and pectin modification genes showed high correlations with specific fiber properties in an inter-specific cotton recombinant inbred line (RIL) population, supporting a role in determining fiber quality.

Morphology of rsw1, a cellulose deficient mutant of Arabidopsis thaliana

SummaryTherswl mutant ofArabidopsis thaliana is mutated in a gene encoding a cellulose synthase catalytic subunit. Mutant seedlings produce almost as much cellulose as the wild type at 21 °C but only about half as much as the wild type at 31 °C. We used this conditional phenotype to investigate how reduced cellulose production affects growth and morphogenesis in various parts of the plant. Roots swell in all tissues at 31 °C, and temperature changes can repeatedly switch them between swollen and slender growth patterns. Dark-grown hypocotyls also swell, whereas cotyledons and rosette leaf blades are smaller, their surfaces are more irregular and their petioles shorter. Leaf trichomes swell and branch abnormally. Plants readily initiate inflorescences at 31 °C which have shorter but not fatter bolts and stomata which bulge above the uneven surface of internodes. Bolts carry the normal number of flowers, but their stigmas protrude beyond the shortened sepals and petals. Anthers dehisce normally, but self-fertilisation is reduced because the stigma is well above the anthers. Anther filaments are short and show a crumpled surface. Viable pollen develops, but female reproductive competence and postpollination development are severely impaired. We conclude that theRSW1 gene is important for cellulose synthesis in many parts of the plant and that reduced cellulose synthesis suppresses organ expansion rather than organ initiation, causes radial swelling only in the root and hypocotyl, but makes the surfaces of many organs uneven. We discuss some possible reasons to explain why different organs vary in their responses. The morphological changes suggest that RSW1 contributes cellulose to primary walls but do not yet exclude a role during secondary-wall deposition.

Arabidopsis dynamin-like protein DRP1A: a null mutant with widespread defects in endocytosis, cellulose synthesis, cytokinesis, and cell expansion

Dynamin-related proteins are large GTPases that deform and cause fission of membranes. The DRP1 family of Arabidopsis thaliana has five members of which DRP1A, DRP1C, and DRP1E are widely expressed. Likely functions of DRP1A were identified by studying rsw9, a null mutant of the Columbia ecotype that grows continuously but with altered morphology. Mutant roots and hypocotyls are short and swollen, features plausibly originating in their cellulose-deficient walls. The reduction in cellulose is specific since non-cellulosic polysaccharides in rsw9 have more arabinose, xylose, and galactose than those in wild type. Cell plates in rsw9 roots lack DRP1A but still retain DRP1E. Abnormally placed and often incomplete cell walls are preceded by abnormally curved cell plates. Notwithstanding these division abnormalities, roots and stems add new cells at wild-type rates and organ elongation slows because rsw9 cells do not grow as long as wild-type cells. Absence of DRP1A reduces endocytotic uptake of FM4-64 into the cytoplasm of root cells and the hypersensitivity of elongation and radial swelling in rsw9 to the trafficking inhibitor monensin suggests that impaired endocytosis may contribute to the development of shorter fatter roots, probably by reducing cellulose synthesis.

A novel isoform of sucrose synthase is targeted to the cell wall during secondary cell wall synthesis in cotton fiber.

Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars.

In Trifolium subterraneum, chalcone synthase is encoded by a ultigene family

Chalcone synthase (CHS) catalyzes the first and key regulatory step in flavonoid biosynthesis. We report the existence and characterization of a CHS multigene family present in Trifolium subterraneum L. cultivar Karridale. The CHS family consists of at least four members, which are tightly clustered in a 15-kb region. The complete sequences of two of these genes (CHS1 and CHS2) are presented. The putative promoters of these genes have sequences which are homologous to those known, or implicated, in regulation of the expression of phenylpropanoid-encoding genes.

Applications of seaweed extracts in Australian agriculture: past, present and future

A rapidly growing world population has highlighted the need to significantly increase food production in the context of a world with accelerating soil and water shortages as well as climatic stressors. This situation has generated new interest in the application of liquid seaweed extracts because of their potent plant growth-enhancing properties through metabolic benefits, triggering disease response pathways and increasing stress tolerance. The basis for these benefits is complex and poorly understood. Liquid seaweed extracts are complex and have been demonstrated to possess novel mechanisms for increasing crop productivity. The benefits of seaweed extracts to crops have previously been reviewed in the context of the northern hemisphere, but not in the context of Australia, its crops and unique stressors. This review considers the application of seaweed extracts in Australian agriculture by (i) introducing the history of the Australian liquid seaweed extract industry and (ii) focusing on evidence of Australian research related to seaweed extract composition, plant growth properties during plant establishment, pathogenic disease and new approaches to phenotyping the biological efficacy of seaweed extracts. This type of research is essential for future Australian agriculture to develop effective strategies for the use of liquid seaweed extracts.

Nucleotide sequence of additional members of the gene family encoding chalcone synthase in Trifolium subterraneum.

CHS (EC 2.3.1.74) catalyzes the condensation of three molecules of malonyl-COA with one molecule of 4-coumaroyl COA to produce 2’,4,4’,6-tetrahydroxychalcone (Heller and Hahlbrock, 1980). The CHS genes have been isolated and characterized in a variety of plants. In the Leguminosae, CHS is a multiple gene family, with subterranean clover (Trifolium subterraneum) having at least nine copies (Arioli et al., 1994). We have recently shown that different copies within this gene family are induced by wounding and Rhizobium infection (Lawson et al., 1994). Screening of a T. subterraneum genomic library with the cDNA clone for CHSZ from bean (Ryder et al., 1987) isolated a number of clones; of these, CHSZ and CHS2 were fully characterized (Arioli et al., 1994). To permit identification of the promoters responsible for the expression of separate gene copies, we determined sequences for additional CHS genes. Sequence data for CHS3 and CHS4, which are linked to CHSZ and CHS2, and CHS5 and CHS6, which, together with a pseudogene CHS7, form a second linkage group, are presented here (Table I). An as-yet unlinked gene, CHS8, has been cloned and partially sequenced to confirm its homology to CHS; this sequence is not presented. The COOH-terminal portion of CHS4 is not located on any of the A clones characterized; thus, the sequence for this gene is truncated 236 bp prior to the end of the gene. Six hundred base pairs of upstream sequence is included in the data base listings for each of these genes. A11 of the CHS sequences within each linkage cluster are oriented in the same direction. The average homology of the six clover CHS genes is 93% at the DNA and 98% at the amino acid level. Curiously, greater homology is present between some members of the two linkage clusters than within each cluster. The phylogenetic relationships be-

Myosins from angiosperms, ferns, and algae amplification of gene fragments with versatile PCR primers and detection of protein products with a monoclonal antibody to a conserved head epitope

SummaryMyosins providing the motors for the actin-based motility that occurs in diverse plants have proved difficult to study. To facilitate those studies, we describe polymerase chain reaction primers that reliably amplify part of the myosin head from diverse plants, consensus sequences that characterise the amplified product as encoding a class V or class VIII myosin, and a monoclonal antibody that recognises an epitope conserved in the head of most plant, fungal, and animal myosins. A pair of stringent oligonucleotide primers was designed that, when used in the polymerase chain reaction, amplified at least eleven different myosins from five species of angiosperms and one sequence from each of the fernAzolla and the algaeNitella andPhaeodactylum. The amplified products, comprising 126 to 135 nucleotides encoding part of the myosin head domain, can be used as myosin-specific probes to screen genomic and cDNA libraries. To identify the products of plant myosin genes, we raised a monoclonal antibody (anti-CHE) to a nine amino acid peptide matching a conserved head epitope showing not more than single amino acid substitutions in most published myosin genes. This antibody recognises rabbit skeletal myosin and multiple polypeptides of >100 kDa in four angiosperms and in the algaNitella. Relating the Mr values of immunoreactive bands inArabidopsis extracts to the predicted Mr values of the products of five myosin genes supports the view that the antibody recognises both myosins V and VIII together with the products of some as yet unsequenced genes. The previously described MB170 antibodies may, in contrast, be specific for one or more type V myosins. Together, the polymerase chain reaction primers and the antibody represent versatile tools for identifying and categorising myosins in diverse plants.