Peter M. Chandler

A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn

Several cDNAs related to an ABA-induced cDNA from barley aleurone were isolated from barley and corn seedlings that were undergoing dehydration. Four different barley polypeptides with sizes of 22.6, 16.2, 14.4 and 14.2 kDa and a single corn polypeptide with a size of 17.0 kDa were predicted from the nucleotide sequences of the cDNAs. These dehydration-induced proteins (dehydrins) are very similar to each other and to a previously identified rice protein induced by ABA and salt, and have at least some similarity to a previously identified cotton embryo protein. Each dehydrin is extremely hydrophilic, glycine-rich, cysteine- and tryptophan-free and contains repeated units in a conserved linear order. A lysine-rich repeating unit occurs twice in each protein, once at the carboxy terminus and once partway through the polypeptide, adjacent to a succession of serines. This repeating unit and the adjacent flanking run of serines are conserved with minimal variation among all dehydrins. Another repeating unit is flanked by the two copies of the lysine-rich unit, and varies in number from one to five copies. This latter repeating unit is less conserved than the former, varying even within a singly dehydrin. The messenger RNAs corresponding to each cDNA are abundant in dehydrating, but not in well-watered seedlings. The amino acid sequence of tryptic peptides from purified dehydration-induced proteins of corn established that the corn cDNAs correspond to a protein that is produced in abundance during the response of corn seedlings to dehydration.

Semidwarf (sd-1), “green revolution” rice, contains a defective gibberellin 20-oxidase gene

The introduction of semidwarf rice (Oryza sativa L.) led to record yield increases throughout Asia in the 1960s. The major semidwarfing allele, sd-1, is still extensively used in modern rice cultivars. The phenotype of sd-1 is consistent with dwarfism that results from a deficiency in gibberellin (GA) plant growth hormones. We propose that the semidwarf (sd-1) phenotype is the result of a deficiency of active GAs in the elongating stem arising from a defective 20-oxidase GA biosynthetic enzyme. Sequence data from the rice genome was combined with previous mapping studies to locate a putative GA 20-oxidase gene (Os20ox2) at the predicted map location of sd-1 on chromosome 1. Two independent sd-1 alleles contained alterations within Os20ox2: a deletion of 280 bp within the coding region of Os20ox2 was predicted to encode a nonfunctional protein in an indica type semidwarf (Doongara), whereas a substitution in an amino acid residue (Leu-266) that is highly conserved among dioxygenases could explain loss of function of Os20ox2 in a japonica semidwarf (Calrose76). The quantification of GAs in elongating stems by GC-MS showed that the initial substrate of GA 20-oxidase activity (GA53) accumulated, whereas the content of the major product (GA20) and of bioactive GA1 was lower in semidwarf compared with tall lines. We propose that the Os20ox2 gene corresponds to the sd-1 locus.

Mutants at the Slender1 Locus of Barley cv Himalaya. Molecular and Physiological Characterization

A dominant dwarf mutant of barley (Hordeum vulgare) that resembles dominant gibberellin (GA) “-insensitive” or “-nonresponsive” mutants in other species is described. α-Amylase production by endosperm half-grains of the mutant required GA3 at concentrations about 100 times that of the WT. The mutant showed only a slight growth response to GA3, even at very high concentrations. However, when additionally dwarfed, growth rate responded to GA3over the normal concentration range, although only back to the original (dwarf) elongation rate. Genetic studies indicated that the dominant dwarf locus was either closely linked or identical to theSln1 (Slender1) locus. A barley sequence related to Arabidopsis GAI/RGA was isolated, and shown to represent the Sln1 locus by the analysis ofsln1 mutants. The dominant dwarf mutant was also altered in this sequence, indicating that it too is an allele atSln1. Thus, mutations at Sln1 generate plants of radically different phenotypes; either dwarfs that are largely dominant and GA “-insensitive/-nonresponsive,” or the recessive slender types in which GA responses appear to be constitutive. Immunoblotting studies showed that in growing leaves, SLN1 protein localized almost exclusively to the leaf elongation zone. In mutants at the Sln1 locus, there were differences in both the abundance and distribution of SLN1 protein, and large changes in the amounts of bioactive GAs, and of their metabolic precursors and catabolites. These results suggest that there are dynamic interactions between SLN1 protein and GA content in determining leaf elongation rate.

Gibberellin Signaling in Barley Aleurone Cells. Control of SLN1 and GAMYB Expression

We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of α-amylase gene expression, in aleurone cells of barley (Hordeum vulgare). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1(Slender1) locus, is necessary for repression of GAMYB in barley aleurone cells. The activity of SLN1 in aleurone cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1, and from thegse1 mutant (that affects GA “sensitivity”), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

SummaryTwo cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA3) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for α-amylase (1,4-α-D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of α-amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the α-amylase clone.Both clones hybridize to RNAs that are similar in size (∼1500b), but which accumulate to different extents following GA3 treatment: α-amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA3 is largely, but not entirely, abolished. These results suggest that GA3 and ABA regulate synthesis of α-amylase in barley aleurone layers primarily through the accumulation of α-amylase mRNA.

Manipulation of plant innate immunity and gibberellin as factor of compatibility in the mutualistic association of barley roots with Piriformospora indica

Fungi of the order Sebacinales (Basidiomycota) are involved in a wide spectrum of mutualistic symbioses with various plants, thereby exhibiting unique potential for biocontrol strategies. Piriformospora indica, a model organism of this fungal order, is able to increase the biomass and grain yield of crop plants, and induces local and systemic resistance to fungal diseases and tolerance to abiotic stress. To elucidate the molecular basis for root colonization, we characterized the interaction of P. indica with barley roots by combining global gene expression profiling, metabolic profiling, and genetic studies. At the metabolic level, we show that fungal colonization reduces the availability of free sugars and amino acids to the root tip. At the transcriptional level, consecutive interaction stages covering pre-penetration-associated events and progressing through to root colonization showed differential regulation of signal perception and transduction components, secondary metabolism, and genes associated with membrane transport. Moreover, we observed stage-specific up-regulation of genes involved in phytohormone metabolism, mainly encompassing gibberellin, auxin and abscisic acid, but salicylic acid-associated gene expression was suppressed. The changes in hormone homoeostasis were accompanied with a general suppression of the plant innate immune system. Further genetic studies showed reduced fungal colonization in mutants that are impaired in gibberellin synthesis as well as perception, and implicate gibberellin as a modulator of the roots basal defence. Our data further reveal the complexity of compatibility mechanisms in host-microbe interactions, and identify gibberellin signaling as potential target for successful fungi.

The effect of different height reducing genes on the early growth of wheat

Genes that reduce height without compromising seedling vigour or coleoptile length have great potential for wheat improvement. We therefore investigated the effects of various reduced height (Rht) genes on the early stages of plant development, using a combination of near isogenic, recombinant, mutant and wild type comparisons. Gibberellin (GA) insensitivity caused by Rht-B1b or Rht-D1b was associated with reduced leaf elongation rate and coleoptile length. Similar results were found for two other sources of dwarfing, Rht11 and Rht17. We found one class of Rht genes (e.g. Rht8) which had no effect on coleoptile length, leaf elongation rate or responsiveness to GA, indicating that these dwarfing genes may act later in wheat development to reduce height and increase harvest index, without affecting early growth. A third class of Rht genes was found in three durum backgrounds. These had reduced coleoptile lengths and leaf elongation rates, but had a greater response to GA than the corresponding tall varieties. We discuss these results in relation to the possible mechanisms underlying the reduction in height and the suitability of the different Rht genes for wheat improvement.

Molecular Characterization of Rht-1 Dwarfing Genes in Hexaploid Wheat

The introduction of the Reduced height (Rht)-B1b and Rht-D1b semidwarfing genes led to impressive increases in wheat (Triticum aestivum) yields during the Green Revolution. The reduction in stem elongation in varieties containing these alleles is caused by a limited response to the phytohormone gibberellin (GA), resulting in improved resistance to stem lodging and yield benefits through an increase in grain number. Rht-B1 and Rht-D1 encode DELLA proteins, which act to repress GA-responsive growth, and their mutant alleles Rht-B1b and Rht-D1b are thought to confer dwarfism by producing more active forms of these growth repressors. While no semidwarfing alleles of Rht-A1 have been identified, we show that this gene is expressed at comparable levels to the other homeologs and represents a potential target for producing novel dwarfing alleles. In this study, we have characterized additional dwarfing mutations in Rht-B1 and Rht-D1. We show that the severe dwarfism conferred by Rht-B1c is caused by an intragenic insertion, which results in an in-frame 90-bp insertion in the transcript and a predicted 30-amino acid insertion within the highly conserved amino-terminal DELLA domain. In contrast, the extreme dwarfism of Rht-D1c is due to overexpression of the semidwarfing Rht-D1b allele, caused by an increase in gene copy number. We show also that the semidwarfing alleles Rht-B1d and Rht-B1e introduce premature stop codons within the amino-terminal coding region. Yeast two-hybrid assays indicate that these newly characterized mutations in Rht-B1 and Rht-D1 confer “GA-insensitive” dwarfism by producing DELLA proteins that do not bind the GA receptor GA INSENSITIVE DWARF1, potentially compromising their targeted degradation.

Identification of a Negative Regulator of Gibberellin Action, HvSPY, in Barley

To broaden our understanding of the molecular mechanisms of gibberellin (GA) action, we isolated a spindly clone (HvSPY) from barley cultivar Himalaya and tested whether the HvSPY protein would modulate GA action in barley aleurone. The HvSPY cDNA showed high sequence identity to Arabidopsis SPY along its entire length, and the barley protein functionally complemented the spy-3 mutation. HvSPY and SPY proteins showed sequence relatedness with animal O-linked N-acetylglucosamine transferases (OGTs), suggesting that they may also have OGT activity. HvSPY has a locus distinct from that of Sln, a mutation that causes the constitutive GA responses of slender barley, which phenotypically resembles Arabidopsis spy mutants. The possibility that the HvSPY gene encodes a negative regulator of GA action was tested by expressing HvSPY in a barley aleurone transient assay system. HvSPY coexpression largely abolished GA3-induced activity of an α-amylase promoter. Surprisingly, HvSPY coexpression increased reporter gene activity from an abscisic acid (ABA)–inducible gene promoter (dehydrin), even in the absence of exogenous ABA. These results show that HvSPY modulates the transcriptional activities of two hormonally regulated promoters: negatively for a GA-induced promoter and positively for an ABA-induced promoter.

Auxin from the Developing Inflorescence Is Required for the Biosynthesis of Active Gibberellins in Barley Stems

Multiple gibberellins (GAs) were quantified in the stems of intact, decapitated, and decapitated auxin-treated barley (Hordeum vulgare) plants. Removal of the developing inflorescence reduced the endogenous levels of indole-3-acetic acid (IAA), GA1, and GA3 and increased the level of GA29 in internodal and nodal tissues below the site of excision. Application of IAA to the excised stump restored GA levels to normal in almost all cases. The conversion of [14C]GA20 to bioactive [14C]GA1 and of [14C]GA5 to bioactive [14C]GA3 was reduced by decapitation, and IAA application was able to restore conversion rates back to the levels found in intact plants. The amount of mRNA for the principal vegetative 3-oxidase (converting GA20 to GA1, and GA5 to GA3) was decreased in decapitated plants and restored by IAA application. The results indicate that the inflorescence of barley is a source of IAA that is transported basipetally into the internodes and nodes where bioactive GA1 and GA3 are biosynthesized. Thus, IAA is required for normal GA biosynthesis in stems, acting at multiple steps in the latter part of the pathway.