Incest versus abstinence: reproductive trade-offs between mate limitation and progeny fitness in a self-incompatible invasive plant.

Plant mating systems represent an evolutionary and ecological trade-off between reproductive assurance through selfing and maximizing progeny fitness through outbreeding. However, many plants with sporophytic self-incompatibility systems exhibit dominance interactions at the S-locus that allow biparental inbreeding, thereby facilitating mating between individuals that share alleles at the S-locus. We investigated this trade-off by estimating mate availability and biparental inbreeding depression in wild radish from five different populations across Australia. We found dominance interactions among S-alleles increased mate availability relative to estimates based on individuals that did not share S-alleles. Twelve of the sixteen fitness variables were significantly reduced by inbreeding. For all the three life-history phases evaluated, self-fertilized offspring suffered a greater than 50% reduction in fitness, while full-sib and half-sib offspring suffered a less than 50% reduction in fitness. Theory indicates that fitness costs greater than 50% can result in an evolutionary trajectory toward a stable state of self-incompatibility (SI). This study suggests that dominance interactions at the S-locus provide a possible third stable state between SI and SC where biparental inbreeding increases mate availability with relatively minor fitness costs. This strategy allows weeds to establish in new environments while maintaining a functional SI system.

Grain dormancy and light quality effects on germination in the model grass Brachypodium distachyon.

• Lack of grain dormancy in cereal crops such as barley and wheat is a common problem affecting farming areas around the world, causing losses in yield and quality because of preharvest sprouting. Control of seed or grain dormancy has been investigated extensively using various approaches in different species, including Arabidopsis and cereals. However, the use of a monocot model plant such as Brachypodium distachyon presents opportunities for the discovery of new genes related to grain dormancy that are not present in modern commercial crops. • In this work we present an anatomical description of the Brachypodium caryopsis, and we describe the dormancy behaviour of six common diploid Brachypodium inbred genotypes. We also study the effect of light quality (blue, red and far-red) on germination, and analyse changes in abscisic acid levels and gene expression between a dormant and a non-dormant Brachypodium genotype. • Our results indicate that different genotypes display high natural variability in grain dormancy and that the characteristics of dormancy and germination are similar to those found in other cereals. • We propose that Brachypodium is an ideal model for studies of grain dormancy in grasses and can be used to identify new strategies for increasing grain dormancy in crop species.

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1), ADPG2, and QUARTET2 are Polygalacturonases required for cell separation during reproductive development in Arabidopsis.

Cell separation is thought to involve degradation of pectin by several hydrolytic enzymes, particularly polygalacturonase (PG). Here, we characterize an activation tagging line with reduced growth and male sterility caused by increased expression of a PG encoded by QUARTET2 (QRT2). QRT2 is essential for pollen grain separation and is part of a small family of three closely related endo-PGs in the Arabidopsis thaliana proteome, including ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1 (ADPG1) and ADPG2. Functional assays and complementation experiments confirm that ADPG1, ADPG2, and QRT2 are PGs. Genetic analysis demonstrates that ADPG1 and ADPG2 are essential for silique dehiscence. In addition, ADPG2 and QRT2 contribute to floral organ abscission, while all three genes contribute to anther dehiscence. Expression analysis is consistent with the observed mutant phenotypes. INDEHISCENT (IND) encodes a putative basic helix-loop-helix required for silique dehiscence, and we demonstrate that the closely related HECATE3 (HEC3) gene is required for normal seed abscission and show that IND and HEC3 are required for normal expression of ADPG1 in the silique dehiscence zone and seed abscission zone, respectively. We also show that jasmonic acid and ethylene act together with abscisic acid to regulate floral organ abscission, in part by promoting QRT2 expression. These results demonstrate that multiple cell separation events, including both abscission and dehiscence, require closely related PG genes.

Functional analysis of HvSPY, a negative regulator of GA response, in barley aleurone cells and Arabidopsis.

SPINDLY (SPY) is an important regulator of plant development, and consists of an N-half tetratricopeptide repeat (TPR) domain containing 10 TPR motifs and a C-half catalytic domain, similar to O-GlcNAc transferase (OGT) of animals. The best characterised role of SPY is a negative regulator of GA signalling, and all known spy alleles have been isolated based on increased GA response. Of the eight alleles that directly affect the TPR domain, all alter TPRs 6, 8 and/or 9. To test the hypothesis that a subset of TPRs, including 6, 8 and 9, are both essential and sufficient for the regulation of GA response, we overexpressed the full-length barley (Hordeum vulgare L.) SPY protein (HvSPY) and several deletion mutants in barley aleurone cells and in Arabidopsis wild type (WT) and spy-4 plants. Transient assays in barley aleurone cells, that also express endogenous HvSPY, demonstrated that introduced HvSPY and HvTPR inhibited GA(3)-induced alpha-amylase expression. With the exception of HvSPYDelta1-5, the other deletion proteins were partially active in the barley assay, including HvSPYDelta6-9 which lacks TPRs 6, 8 and 9. In Arabidopsis, analysis of seed germination under a range of conditions revealed that 35S:HvSPY increased seed dormancy. Hvspy-2, which lacks parts of the eighth and ninth TPRs, was able to partially complement all aspects of the spy-4 phenotype. In the presence of AtSPY, 35S:HvTPR caused some phenotypes consistent with a decrease in GA signalling, including increased seed sensitivity to paclobutrazol and delayed flowering. These plants also possessed distorted leaf morphology and altered epidermal cell shape. Thus, despite genetic analysis demonstrating that TPRs 6, 8 and 9 are required for regulation of GA signalling, our results suggest that these TPRs are neither absolutely essential nor sufficient for SPY activity.

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis.

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

Preliminary development of a genetic strategy to prevent transgene escape by blocking effective pollen flow from transgenic plants.

Genetic modification (GM) of plants has great potential in the production of food and industrial compounds, and in molecular pharming. One of the greatest public concerns regarding this technology is effective pollen flow, in which wind- or insect-borne transgenic pollen is able to fertilise either non-GM crops of the same species, or closely related weed species, and lead to viable seed formation. In this paper we describe a novel concept, based on epigenetic inheritance (imprinting) and post-transcriptional gene silencing (PTGS)/RNA interference (RNAi), designed to prevent transgene escape via pollen flow from transgenic plants. A key advantage of this strategy is that it would allow all seeds from self-pollinated transgenic plants to be harvested and re-sown, without the need for specific treatments, while retaining all of the transgenes present in the parent. Thus, this strategy is not a Genetic Use Restriction Technology (GURT) and if implemented would not prevent seed saving by end-users.

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis.

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.

Functional characterization of AP3, SOC1 and WUS homologues from citrus (Citrus sinensis).

Flowering and flower formation are defining features of angiosperms and the control of these developmental processes involves a common repertoire of genes which are shared among different species of flowering plants. These genes were first identified using various homeotic and flowering time mutants of Arabidopsis and snapdragon, and homologous genes have subsequently been isolated from a wide range of different plant species based on the conservation of protein sequence and function. Using degenerate reverse-transcriptase polymerase chain reaction, we have isolated one APETALA3-like (CitMADS8) and two SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1)-like (CsSL1 and CsSL2) homologues from sweet orange (Citrus sinensis L.). Although the translated amino acid sequence of CitMADS8 shares many similarities with other higher plant APETALA3 proteins, CitMADS8 fails to complement the floral organ identity defects of the Arabidopsis ap3-3 mutant. By contrast, the two citrus SOC1-like genes, particularly CsSL1, are able to shorten the time taken to flower in the Arabidopsis wild-type ecotypes Columbia and C24, and functionally complement the late flowering phenotype of the soc1 mutant, essentially performing the endogenous function of Arabidopsis SOC1. Once flowering has commenced, interactions between specific flowering genes and a gene required for meristem maintenance, WUSCHEL, ensure that the Arabidopsis flower is a determinate structure with four whorls. We have isolated a citrus WUSCHEL homologue (CsWUS) that is capable of restoring most of the meristem function to the shoots and flowers of the Arabidopsis wus-1 mutant, implying that CsWUS is the functional equivalent of Arabidopsis WUSCHEL.

Localised and non-localised promotion of fruit development by seeds in Arabidopsis.

In Arabidopsis, as in the majority of flowering plants, developing seeds promote fruit growth. One method to investigate this interaction is to use plants with reduced seed set and determine the effect on fruit growth. Plants homozygous for a transgene designed to ectopically express a gene encoding a gibberellin-deactivating enzyme exhibit reduced pollen tube elongation, suggesting that the plant hormone gibberellin is required for this process. Reduced pollen tube growth causes reduced seed set and decreased silique (fruit) size, and this genotype is used to explore the relationship between seed set and fruit elongation. A detailed analysis of seed set in the transgenic line reveals that reduced pollen tube growth decreases the probability of each ovule being fertilised. This effect becomes progressively more severe as the distance between the stigma and the ovule increases, revealing the complex biology underlying seed fertilisation. In terms of seed-promoted fruit growth, major localised and minor non-localised components that contribute to final silique length can be identified. This result demonstrates that despite the relatively small size of the fruit and associated structures, Arabidopsis can be used as a model to investigate fundamental questions in fruit physiology.

Regulation of the early GA biosynthesis pathway in pea.

The early steps in the gibberellin (GA) biosynthetic pathway are controlled by single copy genes or small gene families. In pea (Pisum sativum L.) there are two ent-kaurenoic acid oxidases, one expressed only in the seeds, while ent-copalyl synthesis and ent-kaurene oxidation appear to be controlled by single copy genes. None of these genes appear to show feedback regulation and the only major developmental regulation appears to be during seed development. During shoot maturation, transcript levels do not change markedly with the result that all the three genes examined are expressed in mature tissue, supporting recent findings that these tissues can synthesise GAs. It therefore appears that the regulation of bioactive GA levels are determined by the enzymes encoded by the 2-oxoglutarate-dependent dioxygenase gene families controlling the later steps in GA biosynthesis. However the early steps are nonetheless important as a clear log/linear relationship exists between elongation and the level of GA1 in a range of single and double mutants in genes controlling these steps.

Tall tales from sly dwarves: novel functions of gibberellins in plant development.

Gibberellins (GAs) are endogenous hormones controlling numerous aspects of plant growth and development. Our present understanding of GA physiology is based largely on genetic analysis in model plants such as Arabidopsis. In spite of the success of this approach, the discovery of additional physiological roles for GAs in seed development, pollen tube growth and meristem development indicates that the existing collection of GA-related mutants (identified partially or entirely on the basis of vegetative phenotypes) has failed to uncover all aspects of plant development that are controlled by GAs. The continued use of ever improving forward and reverse genetic techniques is expected to lead to the discovery of further novel roles for GAs in plant development.

Plants with increased expression of ent-kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

The gibberellin (GA) biosynthetic pathway includes the three-step oxidation of ent-kaurene to ent-kaurenoic acid, catalyzed by the enzyme ent-kaurene oxidase (KO). Arabidopsis plants overexpressing the KO cDNA under the control of the cauliflower mosaic virus 35S promoter, with or without a translational fusion to a modified green fluorescent protein (GFP), are very similar to wild-type (WT) plants under normal growth conditions. In contrast, when WT and 35S:KO (or 35S:KO-GFP) seeds, seedlings or pollen tubes are grown in the presence of chemical inhibitors of KO, such as paclobutrazol and uniconazole, plants with increased KO expression are partially resistant to the effects of these inhibitors. In combination with the observation that decreased KO levels increase the sensitivity to KO inhibitors, the 35S:KO phenotypes demonstrate that the modification of KO enzyme levels could be used to create transgenic crop plants with altered KO inhibitor response. These results also suggest that the KO gene could be used as a selectable marker for plant regeneration based on resistance to KO inhibitors. Finally, the observation that pollen tubes expressing 35S:KO or 35S:KO-GFP have decreased sensitivity to KO inhibitors provides further evidence for a physiological role for GAs in pollen tube elongation.

The gar2 and rga alleles increase the growth of gibberellin-deficient pollen tubes in Arabidopsis.

Ectopic expression in Arabidopsis of a pea (Pisum sativum) cDNA (2ox2) encoding a gibberellin (GA) 2-oxidase (PsGA2ox2), involved in the deactivation of biologically active GAs, has been used to establish a role for GAs in promoting pollen tube growth. One line, 35S:2ox2/28c, when homozygous for the transgene, exhibits a novel small fruit phenotype. The 28c transgene reduces pollen tube growth, and this results in a reduced number of fertilized seeds that are only present at the end of the silique nearest the stigma. To confirm that the 28c pollen tube phenotype is due to sense expression of the 2ox2 mRNA, a "hairpin" RNA interface silencing construct, designed to silence 2ox2 expression, has been used to restore pollen tube growth and fruit development. The interaction between 28c and other mutants with increased GA response has also been examined to provide further evidence that GAs play an important role in pollen tube growth. Based on the ability of mutant alleles to suppress the 35S:2ox2/28c phenotype, we define new roles for the gar2-1 and rga alleles in GA signaling during pollen tube elongation in addition to their previously established roles in vegetative tissues. In contrast to the constitutive GA response observed in internodes and leaves lacking RGA and GAI, the rga-2 gai-d5 mutant combination is only a partial suppressor of the 28c phenotype. Because the dominant dwarfing gai-1 allele reduces GA response in vegetative tissues, its effect on plant fertility has been examined. Although gai-1 reduces seed set, this appears to reflect defects in reproductive development other than pollen tube function. Finally, we show that the genetic background (Landsberg erecta or Columbia) modifies the 28c phenotype and that this effect is not due to the ER/er difference between these two ecotypes.

Gibberellins are required for seed development and pollen tube growth in Arabidopsis.

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.

SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant.

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.