Abstract. We have isolated and sequenced a fulllength cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with ot tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 Mr intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000Mr intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH2-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in proteinprotein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.

We have used an insertional mutagenesis/ gene tagging technique to generate new Chlamydomonas reinhardtii mutants that are defective in assembly of the outer dynein arm. Among 39 insertional oda mutants characterized, two are alleles of the previously uncloned ODA3 gene, one is an allele of the uncloned ODA10 gene, and one represents a novel ODA gene (termed ODA12). ODA3 is of particular interest because it is essential for assembly of both the outer dynein arm and the outer dynein arm docking complex (ODA-DC) onto flagellar doublet microtubules (Takada, S., and R. Kamiya. 1994. J. Cell Biol. 126:737– 745). Beginning with the inserted DNA as a tag, the ODA3 gene and a full-length cDNA were cloned. The cloned gene rescues the phenotype of oda3 mutants. The cDNA sequence predicts a novel 83.4-kD protein with extensive coiled-coil domains. The ODA-DC contains three polypeptides; direct amino acid sequencing indicates that the largest of these polypeptides corresponds to ODA3. This protein is likely to have an important role in the precise positioning of the outer dynein arms on the flagellar axoneme.

We projected meta-QTL (MQTL) for drought, salinity, and waterlogging tolerance to the physical map of barley through meta-analysis. The positions of these MQTL were refined and candidate genes were identified. Drought, salinity and waterlogging are three major abiotic stresses limiting barley yield worldwide. Breeding for abiotic stress-tolerant crops has drawn increased attention, and a large number of quantitative trait loci (QTL) for drought, salinity, and waterlogging tolerance in barley have been detected. However, very few QTL have been successfully used in marker-assisted selection (MAS) in breeding. In this study, we summarized 632 QTL for drought, salinity and waterlogging tolerance in barley. Among all these QTL, only 195 major QTL were used to conduct meta-analysis to refine QTL positions for MAS. Meta-analysis was used to map the summarized major QTL for drought, salinity, and waterlogging tolerance from different mapping populations on the barley physical map. The positions of identified meta-QTL (MQTL) were used to search for candidate genes for drought, salinity, and waterlogging tolerance in barley. Both MQTL3H.4 and MQTL6H.2 control drought tolerance in barley. Fine-mapped QTL for salinity tolerance, HvNax4 and HvNax3, were validated on MQTL1H.4 and MQTL7H.2, respectively. MQTL2H.1 and MQTL5H.3 were also the target regions for improving salinity tolerance in barley. MQTL4H.4 is the main region controlling waterlogging tolerance in barley with fine-mapped QTL for aerenchyma formation under waterlogging conditions. Detected and refined MQTL and candidate genes are crucial for future successful MAS in barley breeding.

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Cell sizes are linked across multiple tissues, including stomata, and this variation is closely correlated with genome size. These associations raise the question of whether generic changes in cell size cause suboptimal changes in stomata, requiring subsequent evolution under selection for stomatal size. We tested the relationships among guard cell length, genome size and vegetation type using phylogenetically independent analyses on 67 species of the ecologically and structurally diverse family, Proteaceae. We also compared how genome and stomatal sizes varied at ancient (among genera) and more recent (within genus) levels. The observed 60-fold range in genome size in Proteaceae largely reflected the mean chromosome size. Compared with variation among genera, genome size varied much less within genera (< 6% of total variance) than stomatal size, implying evolution in stomatal size subsequent to changes in genome size. Open vegetation and closed forest had significantly different relationships between stomatal and genome sizes. Ancient changes in genome size clearly influenced stomatal size in Proteaceae, but adaptation to habitat strongly modified the genome–stomatal size relationship. Direct adaptation to the environment in stomatal size argues that new proxies for past concentrations of atmospheric CO2 that incorporate stomatal size are superior to older models based solely on stomatal frequency.

Aerenchyma formation after 7 days of waterlogging in commercial potting mixture can be a reliable, fast, and widely utilized approach for the selection of waterlogging tolerant barley genotypes. One major QTL for aerenchyma formation after 7 days of waterlogging treatment was identified and the newly developed markers explained 44 % of the phenotypic variance. This QTL can now be effectively used in barley breeding programs. Waterlogging is one of the important limiting conditions for crop yield and productivity. The main feature of waterlogged soils is oxygen deprivation, due to slow gas diffusion in water. Decreased oxygen content in waterlogged soils leads to the oxygen deficiency in plant tissues, resulting in reduced energy availability for plants. Rapidly induced aerenchyma formation is critical to maintaining adequate oxygen supply and overall waterlogging tolerance in barley. In this study, we have proved that quantifying aerenchyma formation after 7 days of waterlogging in commercial potting mixture can be a reliable, fast, and widely utilised approach for the selection of waterlogging tolerant barley genotypes, which is supported by measurements of redox potential (an indicator of anaerobic conditions). This protocol was also used to identify quantitative trait loci (QTL) in a doubled haploid population of barley from the cross between Yerong (tolerant) and Franklin (sensitive) genotypes. The QTL for aerenchyma formation and root porosity were at the same location as the waterlogging tolerance QTL. Seven new markers were developed and added onto this region on chromosome 4H. One major QTL for aerenchyma formation after 7 days waterlogging treatment explained 44.0 % of the phenotypic variance. This successful QTL for aerenchyma formation can be effectively used in the marker assisted selection to improve waterlogging tolerance in barley.

Background and aims Plant adaptation to waterlogged conditions requires a set of morphological and physiological/biochemical changes. The formation of aerenchyma is one of the most crucial adaptive traits for waterlogging tolerance. Enzymatic scavenging may also potentially contribute to waterlogging tolerance by providing detoxification of reactive oxygen species (ROS). Methods Changes of root porosity (as an indicator of aerenchyma formation) and activities in leaves of four major antioxidant enzymes, γ-amino butyric acid (GABA) and lactic acid contents in roots were evaluated in six barley genotypes contrasting in waterlogging tolerance.Results Soil waterlogging caused significant increases in adventitious root porosity in all genotypes. Waterlogging-tolerant genotypes showed not only significantly higher adventitious root porosity than sensitive genotypes but also much faster development of aerenchyma. The greatest difference in adventitious root porosity among genotypes was observed after 7 days of Plant Soil waterlogging treatment. At the same time, antioxidant enzyme activities in leaves, GABA and lactic acid contents in roots did not correlate with waterlogging tolerance. Conclusions A faster formation of aerenchyma in adventitious roots is one of the key factors for waterlogging tolerance in barley. This protocol is recommended to be applied in future studies to identify molecular markers linked to this trait using appropriate mapping populations.

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