Ecophysiology of seed dormancy in the Australian endemic species Acanthocarpus preissii (Dasypogonaceae)

BACKGROUND AND AIMS: Seedlings of Acanthocarpus preissii are needed for coastal sand dune restoration in Western Australia. However, seeds of this Western Australian endemic have proven to be very difficult to germinate. The aims of this study were to define a dormancy-breaking protocol, identify time of suitable conditions for dormancy-break in the field and classify the type of seed dormancy in this species. METHODS: Viability, water-uptake (imbibition) and seed and embryo characteristics were assessed for seeds collected in 2003 and in 2004 from two locations. The effects of GA(3), smoke-water, GA(3) + smoke-water and warm stratification were tested on seed dormancy-break. In a field study, soil temperature and the moisture content of soil and buried seeds were monitored for 1 year. KEY RESULTS: Viability of fresh seeds was >90 %, and they had a fully developed, curved-linear embryo. Fresh seeds imbibed water readily, with mass increasing approx. 52 % in 4 d. Non-treated fresh seeds and those exposed to 1000 ppm GA(3), 1 : 10 (v/v) smoke-water/water or 1000 ppm GA(3) + 1 : 10 (v/v) smoke-water/water germinated <8 %. Fresh seeds germinated to >80 % when warm-stratified for at least 7 weeks at 18/33 degrees C and then moved to 7/18 degrees C, whereas seeds incubated continuously at 7/18 degrees C germinated to <20 %. CONCLUSIONS: Seeds of A. preisii have non-deep physiological dormancy that is released by a period of warm stratification. Autumn (March/April) is the most likely time for warm stratification of seeds of this species in the field. This is the first report of the requirement for warm stratification for dormancy release in seeds of an Australian species.     

Characterization and mapping of a spotted leaf mutant in rice (Oryza sativa)

Spotted leaf mutant belongs to a class of mutants that can produce necrotic lesions spontaneously in plants without any attack by pathogens. These mutants have no beneficial effect on plant productivity but provide a unique opportunity to study programmed cell death in plant defense responses. A novel rice spotted leaf mutant (spl30) was isolated through low-energy heavy ion irradiation. Lesion expression was sensitive to light and humidity. The spl30 mutant caused a decrease in chlorophyll and soluble protein content, with marked accumulation of reactive oxygen species (ROS) around the lesions. In addition, the spl30 mutant significantly enhanced resistance to rice bacterial blight (X. oryzae pv. oryzae) from China (C1–C7). The use of SSR markers showed that the spl30 gene was located between markers XSN2 and XSN4. The genetic distance between the spl30 gene and XSN2 and between spl30 and XSN4 was 1.7 cM and 0.2 cM, respectively. The spl30 gene is a new gene involved in lesion production and may be related to programmed cell death in rice. The ability of this mutant to confer broad resistance to bacterial blight provides a model for studying the interaction between plants and pathogenic bacteria.     

Carbon fluxes in the rhizosphere of sweet chestnut seedlings (Castanea sativa) grown under two atmospheric CO2 concentrations: 14C partitioning after pulse labelling

Partitioning of ¹⁴C was assessed in sweet chestnut seedlings (Castanea sativa Mill.) grown in ambient and elevated atmospheric [CO₂] environments during two vegetative cycles. The seedlings were exposed to ¹⁴CO₂ atmosphere in both high and low [CO₂] environments for a 6-day pulse period under controlled laboratory conditions. Six days after exposure to ¹⁴CO₂, the plants were harvested, their dry mass and the radioactivity were evaluated. ¹⁴C concentration in plant tissues, root-soil system respiratory outputs and soil residues (rhizodeposition) were measured. Root production and rhizodeposition were increased in plants growing in elevated atmospheric [CO₂]. When measuring total respiration, i.e. CO₂ released from the root/soil system, it is difficult to separate CO₂ originating from roots and that coming from the rhizospheric microflora. For this reason a model accounting for kinetics of exudate mineralization was used to estimate respiration of rhizospheric microflora and roots separately. Root activity (respiration and exudation) was increased at the higher atmospheric CO₂ concentration. The proportion attributed to root respiration accounted for 70 to 90% of the total respiration. Microbial respiration was related to the amount of organic carbon available in the rhizosphere and showed a seasonal variation dependent upon the balance of root exudation and respiration. The increased carbon assimilated by plants grown under elevated atmospheric [CO₂] stayed equally distributed between these increased root activities. ei]H Lambers     

Expressed sequence tag analysis of khat (Catha edulis) provides a putative molecular biochemical basis for the biosynthesis of phenylpropylamino alkaloids

Khat (Catha edulis Forsk.) is a flowering perennial shrub cultivated for its neurostimulant properties resulting mainly from the occurrence of (S)-cathinone in young leaves. The biosynthesis of (S)-cathinone and the related phenylpropylamino alkaloids (1S,2S)-cathine and (1R,2S)-norephedrine is not well characterized in plants. We prepared a cDNA library from young khat leaves and sequenced 4,896 random clones, generating an expressed sequence tag (EST) library of 3,293 unigenes. Putative functions were assigned to > 98% of the ESTs, providing a key resource for gene discovery. Candidates potentially involved at various stages of phenylpropylamino alkaloid biosynthesis from L-phenylalanine to (1S,2S)-cathine were identified.     

Co-adaptation of seed dormancy and flowering time in the arable weed Capsella bursa-pastoris (shepherd's purse)

Background and Aims

The duration of the plant life cycle is an important attribute that determines fitness and coexistence of weeds in arable fields. It depends on the timing of two key life-history traits: time from seed dispersal to germination and time from germination to flowering. These traits are components of the time to reproduction. Dormancy results in reduced and delayed germination, thus increasing time to reproduction. Genotypes in the arable seedbank predominantly have short time to flowering. Synergy between reduced seed dormancy and reduced flowering time would create stronger contrasts between genotypes, offering greater adaptation in-field. Therefore, we studied differences in seed dormancy between in-field flowering time genotypes of shepherd''s purse.

Methods

Genotypes with early, intermediate or late flowering time were grown in a glasshouse to provide seed stock for germination tests. Secondary dormancy was assessed by comparing germination before and after dark-incubation. Dormancy was characterized separately for seed myxospermy heteromorphs, observed in each genotype. Seed carbon and nitrogen content and seed mass were determined as indicators of seed filling and resource partitioning associated with dormancy.

Key Results

Although no differences were observed in primary dormancy, secondary dormancy was weaker among the seeds of early-flowering genotypes. On average, myxospermous seeds showed stronger secondary dormancy than non-myxospermous seeds in all genotypes. Seed filling was similar between the genotypes, but nitrogen partitioning was higher in early-flowering genotypes and in non-myxospermous seeds.

Conclusions

In shepherd''s purse, early flowering and reduced seed dormancy coincide and appear to be linked. The seed heteromorphism contributes to variation in dormancy. Three functional groups of seed dormancy were identified, varying in dormancy depth and nitrate response. One of these groups (FG-III) was distinct for early-flowering genotypes. The weaker secondary dormancy of early-flowering genotypes confers a selective advantage in arable fields.     

In vivo putative O-GlcNAcylation of human SCP1 and evidence for possible role of its N-terminal disordered structure

RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are responsible for the dephosphorylation of the C-terminal domain of the small subunit of RNAPII in eukaryotes. Recently, we demonstrated the identification of several interacting partners with human small CTD phosphatase1 (hSCP1) and the substrate specificity to delineate an appearance of the dephosphorylation catalyzed by SCP1. In this study, using the established cells for inducibly expressing hSCP1 proteins, we monitored the modification of β-O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation is one of the most common post-translational modifications (PTMs). To gain insight into the PTM of hSCP1, we used the Western blot, immunoprecipitation, succinylayed wheat germ agglutininprecipitation, liquid chromatography-mass spectrometry analyses, and site-directed mutagenesis and identified the Ser⁴¹ residue of hSCP1 as the O-GlcNAc modification site. These results suggest that hSCP1 may be an O-GlcNAcylated protein in vivo, and its N-terminus may function a possible role in the PTM, providing a scaffold for binding the protein(s). [BMB Reports 2014; 47(10): 593-598]     

Sympatric species of Hibbertia (Dilleniaceae) vary in dormancy break and germination requirements: implications for classifying morphophysiological dormancy in Mediterranean biomes

Background and Aims

Several ecologically important plant families in Mediterranean biomes have seeds with morphophysiological dormancy (MPD) but have been poorly studied. The aim of this study was to understand the seed ecology of these species by focusing on the prominent, yet intractably dormant Australian genus Hibbertia. It was hypothesized that the slow germination in species of this genus is caused by a requirement for embryo growth inside the seed before germination, and that initiation of embryo growth is reliant upon a complex sequence of environmental cues including seasonal fluctuations in temperature and moisture, and an interplay with light and smoke. Using the results, the classification of the MPD level in species of Hibbertia is considered.

Methods

Four species of Hibbertia in winter rainfall south-western Australia were selected. These species, whilst differing in geographic distributions, are variously sympatric, and all are important understorey components of plant communities. The following aspects related to dormancy break, embryo growth and germination were investigated: temperature and moisture requirements; effects of karrikinolide, gibberellic acid and aerosol smoke; and phenology.

Key Results

Following exposure to wet/dry cycles at low or high temperatures, embryo growth and germination occurred, albeit slowly in all species at low temperatures when moisture was unlimited, corresponding to winter in south-west Australia. Photo regime influenced germination only in H. racemosa. Aerosol smoke triggered substantial germination during the 1st germination season in H. huegelii and H. hypericoides.

Conclusions

Although the study species are con-generic, sympatric and produce seeds of identical morphology, they possessed different dormancy-break and germination requirements. The physiological component of MPD was non-deep in H. racemosa but varied in the other three species where more deeply dormant seeds required >1 summer to overcome dormancy and, thus, germination was spread over time. Embryos grew during winter, but future studies need to resolve the role of cold versus warm stratification by using constant temperature regimes. To include Mediterranean species with MPD, some modifications to the current seed-dormancy classification system may need consideration: (a) wet/dry conditions for warm stratification and (b) a relatively long period for warm stratification. These outcomes have important implications for improving experimental approaches to resolve the effective use of broadcast seed for ecological restoration.     

Acquisition of physical dormancy and ontogeny of the micropyle--water-gap complex in developing seeds of Geranium carolinianum (Geraniaceae)

Background and Aims

The ‘hinged valve gap’ has been previously identified as the initial site of water entry (i.e. water gap) in physically dormant (PY) seeds of Geranium carolinianum (Geraniaceae). However, neither the ontogeny of the hinged valve gap nor acquisition of PY by seeds of Geraniaceae has been studied previously. The aims of the present study were to investigate the physiological events related to acquisition of PY and the ontogeny of the hinged valve gap and seed coat of G. carolinianum.

Methods

Seeds of G. carolinianum were studied from the ovule stage until dispersal. The developmental stages of acquisition of germinability, physiological maturity and PY were determined by seed measurement, germination and imbibition experiments using intact seeds and isolated embryos of both fresh and slow-dried seeds. Ontogeny of the seed coat and water gap was studied using light microscopy.

Key Results

Developing seeds achieved germinability, physiological maturity and PY on days 9, 14 and 20 after pollination (DAP), respectively. The critical moisture content of seeds on acquisition of PY was 11 %. Slow-drying caused the stage of acquisition of PY to shift from 20 to 13 DAP. Greater extent of cell division and differentiation at the micropyle, water gap and chalaza than at the rest of the seed coat resulted in particular anatomical features. Palisade and subpalisade cells of varying forms developed in these sites. A clear demarcation between the water gap and micropyle is not evident due to their close proximity.

Conclusions

Acquisition of PY in seeds of G. carolinianum occurs after physiological maturity and is triggered by maturation drying. The micropyle and water gap cannot be considered as two separate entities, and thus it is more appropriate to consider them together as a ‘micropyle–water-gap complex’.     

Modeling and phylogenetic analysis of cytosolic ascorbate peroxidase (OsAPX1) from rice reveal signature motifs that may play a role in stress tolerance

Ascorbate peroxidase (APX) is a crucial, haeme-containing enzyme of the ascorbate glutathione cycle that detoxifies reactive oxygen species in plants by catalyzing the conversion of hydrogen peroxide to water using ascorbate as a specific electron donor. Different APX isoforms are present in discrete subcellular compartments in rice and their expression is stress regulated. We revealed the homology model of OsAPX1 protein using the crystal structure of soybean GmAPX1 (PDB ID: 2XIF) as template by Modeller 9.12. The resultant OsAPX1 model structure was refined by PROCHECK, ProSA, Verify3D and RMSD that indicated the model structure is reliable with 83 % amino acid sequence identity with template, RMSD (1.4 Å), Verify3D (86.06 %), Zscores (-8.44) and Ramachandran plot analysis showed that conformations for 94.6% of amino acid residues are within the most favoured regions. Investigation revealed two conserved signatures for haeme ligand binding and peroxidase activity in the alpha helical region that may play a significant role during stress.     

Spatio-temporal relief from hypoxia and production of reactive oxygen species during bud burst in grapevine (Vitis vinifera)

Background and Aims Plants regulate cellular oxygen partial pressures (pO₂), together with reduction/oxidation (redox) state in order to manage rapid developmental transitions such as bud burst after a period of quiescence. However, our understanding of pO₂ regulation in complex meristematic organs such as buds is incomplete and, in particular, lacks spatial resolution.Methods The gradients in pO₂ from the outer scales to the primary meristem complex were measured in grapevine (Vitis vinifera) buds, together with respiratory CO₂ production rates and the accumulation of superoxide and hydrogen peroxide, from ecodormancy through the first 72 h preceding bud burst, triggered by the transition from low to ambient temperatures.Key Results Steep internal pO₂ gradients were measured in dormant buds with values as low as 2·5 kPa found in the core of the bud prior to bud burst. Respiratory CO₂ production rates increased soon after the transition from low to ambient temperatures and the bud tissues gradually became oxygenated in a patterned process. Within 3 h of the transition to ambient temperatures, superoxide accumulation was observed in the cambial meristem, co-localizing with lignified cellulose associated with pro-vascular tissues. Thereafter, superoxide accumulated in other areas subtending the apical meristem complex, in the absence of significant hydrogen peroxide accumulation, except in the cambial meristem. By 72 h, the internal pO₂ gradient showed a biphasic profile, where the minimum pO₂ was external to the core of the bud complex.Conclusions Spatial and temporal control of the tissue oxygen environment occurs within quiescent buds, and the transition from quiescence to bud burst is accompanied by a regulated relaxation of the hypoxic state and accumulation of reactive oxygen species within the developing cambium and vascular tissues of the heterotrophic grapevine buds.     

Molecular Characterization of a Soybean Cyst Nematode (Heterodera glycines) Homolog of unc-87

In Caenorhabditis elegans the unc-87 gene encodes a protein that binds to actin at the I band and is important in nematodes for maintenance of the body-wall muscle. Caenorhabditis elegans mutant phenotypes of unc-87 exhibit severe paralysis in larvae and limp paralysis in the adult. We cloned and characterized a full-length cDNA representing a Heterodera glycines homolog of the unc-87 gene from C. elegans that encodes a protein that contains a region of seven repeats similar to CLIK-23 from C-elegans and has 81% amino acid identity with that of C. elegans unc-87 variant A. In the EST database clones labeled "unc-87'''' encode mainly the 3'' portion of unc-87, while clones labeled "calponin homolog OV9M'''' contain mainly DNA sequence representing the 5'' and middle transcribed regions of unc-87. A 1770 nucleotide cDNA encoding H. glycines unc-87 was cloned and encodes a predicted UNC-87 protein product of 375 amino acids. The expression of unc-87 was determined using RT-PCR and, in comparison to its expression in eggs, unc-87 was expressed 6-fold higher in J2 juveniles and 20-fold and 13-fold (P = 0.05) higher in nematodes 15 and 30 days after inoculation, respectively. In situ hybridization patterns confirmed the expression patterns observed with RT-PCR.     

Molecular cloning and expression analysis of a monosaccharide transporter gene OsMST4 from rice (Oryza sativa L.)

Two putative glycosyltransferases in Arabidopsis thaliana, designated reduced residual arabinose-1 and -2 (RRA1 and RRA2), are characterized at the molecular level. Both genes are classified in CAZy GT-family-77 and are phylogenetically related to putative glycosyltranferases of Chlamydomonas reinhardtii. The expression pattern of the two genes was analyzed by semi-quantitative RT-PCR using mRNA extracted from various organs of bolting Arabidopsis thaliana plants. In addition, promoter::gusA analysis of transgenic Arabidopsis thaliana containing a fusion between either the RRA-1 or -2 promoter fragment and the gusA reporter gene showed that whereas the RRA1 promoter was primarily active in the apical meristem, the expression pattern of the RRA2 promoter was more diverse but also highly active in the meristematic region. In addition, T-DNA mutant insertion lines of both RRA-1 and -2, were identified and characterized at the molecular and biochemical level. Monosaccharide compositional analyses of cell wall material isolated from the meristematic region showed a ca. 20% reduction in the arabinose content in the insoluble/undigested cell wall residue after enzymatic removal of xyloglucan and pectic polysaccharides. These data indicate that both RRA-1 and -2 play a role in the arabinosylation of cell wall component(s).     

Posttranslational regulation of pyruvate, orthophosphate dikinase in developing rice (Oryza sativa) seeds

Pyruvate, orthophosphate dikinase (PPDK; E.C.2.7.9.1) is most well known as a photosynthetic enzyme in C₄ plants. The enzyme is also ubiquitous in C₃ plant tissues, although a precise non-photosynthetic C₃ function(s) is yet to be validated, owing largely to its low abundance in most C₃ organs. The single C₃ organ type where PPDK is in high abundance, and, therefore, where its function is most amenable to elucidation, are the developing seeds of graminaceous cereals. In this report, we suggest a non-photosynthetic function for C₃ PPDK by characterizing its abundance and posttranslational regulation in developing Oryza sativa (rice) seeds. Using primarily an immunoblot-based approach, we show that PPDK is a massively expressed protein during the early syncitial-endosperm/-cellularization stage of seed development. As seed development progresses from this early stage, the enzyme undergoes a rapid, posttranslational down-regulation in activity and amount via regulatory threonyl-phosphorylation (PPDK inactivation) and protein degradation. Immunoblot analysis of separated seed tissue fractions (pericarp, embryo + aleurone, seed embryo) revealed that regulatory phosphorylation of PPDK occurs in the non-green seed embryo and green outer pericarp layer, but not in the endosperm + aleurone layer. The modestly abundant pool of inactive PPDK (phosphorylated + dephosphorylated) that was found to persist in mature rice seeds was shown to remain largely unchanged (inactive) upon seed germination, suggesting that PPDK in rice seeds function in developmental rather than in post-developmental processes. These and related observations lead us to postulate a putative function for the enzyme that aligns its PEP to pyruvate-forming reaction with biosynthetic processes that are specific to early cereal seed development.