A and D genomes spatial separation at somatic metaphase in tetraploid cotton: evidence for genomic disposition in a polyploid plant

Chromosomal dispositions were analyzed on the metaphase plate of tetraploid cotton (AADD). At metaphase, the two subgenomes, A and D, were separated in a radial pattern in which the small D subgenome chromosomes tended to concentrate at the center and the large A subgenome chromosomes were scattered about the periphery on the metaphase plate. Although the ordered chromosome arrangement was disturbed in an artificial hexaploid (AADDGG), the separation pattern could be recovered after the majority of the additional genome (GG) chromosomes were removed by backcrossing the artificial hexaploid with the tetraploid cotton (AADD). A similar genome separation phenomenon was also found in synthesized tetraploid cotton (AAGG). These results indicate that the genome separation pattern could be established immediately after tetraploid cotton formation and could be stably inherited in tetraploid cotton. Given the evidence of parental genome separation in other plants and animals, we speculated that genome separation might be a normal phenomenon in diploid and polyploid species. These finding will shed light on the chromosome conformation in plant cells.     

Soluble carbohydrate content of soybean [Glycine max (L.) merr.] somatic and zygotic embryos during development

Summary Somatic and zygotic embryos of soybean cv. Jack were analyzed for soluble carbohydrate, total lipids, and protein during development. Zygotic embryos accumulated trace amounts of fructose, galactose, and galactinol., whereas somatic embryos contained only trace amounts of galactose. Somatic embryos accumulated much higher glucose levels than zygotic embryos. Both somatic and zygotic embryos contain low levels of sucrose, myoinositol, and pinitol. Raffinose and stachyose accumulated in the late developmental stages of zygotic embryos, but only stachyose was found to accumulate in the late stage somatic embryos. Zygotic embryos contained low total lipid levels up to 50 d after flowering (DAF) and then the levels increased to 16% by 55 DAF and 21% at 65 DAF. Somatic embryos had low levels of total lipids throughout development with the maximum of only 4.7%. Soybean zygotic embryos contained about 40% protein throughout development, while the protein concentration of somatic embryos decreased from 44% to 25% as maturation approached. These studies demonstrate that the composition of Jack zygotic embryos is similar to that described for other cultivars during development while the somatic embryo composition and size is markedly different. The low somatic embryo germination often noted might be due to the abnormal development as shown by a composition different from that of mature zygotic embryos. The low concentration of the raffinose series sugars might be especially important factors.     

Water relations parameters and tissue development in somatic and zygotic embryos of three pinaceous conifers

There is increasing interest in using somatic embryogenesis to meet the demand for high quality seedlings. However, in vitro production of propagules on a large scale depends on the optimization of the maturation and germination steps promoted by desiccation and subsequent imbibition of the embryo, respectively. It is therefore important to characterize zygotic and somatic embryos in terms of their water relations. Bound water, elastic modulus, osmotic potential at full turgor, and relative water content at turgor loss point were determined for somatic and zygotic embryos of western larch and loblolly pine and somatic embryos of white spruce at two developmental stages. These water relations parameters were derived from water-release curves obtained by suspending tissue samples in sealed vials over unsaturated NaCl solutions of known water potential. There was little difference in water relations parameters among species but marked stage dependency for bound water and elastic modulus. The amount of bound water was lowest in western larch somatic embryos (0.02-0.07) and highest in zygotic loblolly pine embryos (0.10-0.18). Elastic modulus ranged from 2.5 to 6.2 MPa in somatic embryos but varied between 1.4 and 1.8 MPa in zygotic embryos. The osmotic potential was lower in somatic embryos than in their zygotic counterparts. Our results show that water relations parameters are remarkably conservative across species but that, within a given species, these parameters are stage specific. It would seem, therefore, that desiccation protocols might be developed for a given developmental stage and applied across a range of species without the confounding effects of differences in water relations parameters.     

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum)

Cultivated cotton (Gossypium hirsutum) is the most important fibre crop in the world. Cotton leaf curl disease (CLCuD) is the major limiting factor and a threat to textile industry in India and Pakistan. All the local cotton cultivars exhibit moderate to no resistance against CLCuD. In this study, we evaluated an exotic cotton accession Mac7 as a resistance source to CLCuD by challenging it with viruliferous whiteflies and performing qPCR to evaluate the presence/absence and relative titre of CLCuD‐associated geminiviruses/betasatellites. The results indicated that replication of pathogenicity determinant betasatellite is significantly attenuated in Mac7 and probably responsible for resistance phenotype. Afterwards, to decipher the genetic basis of CLCuD resistance in Mac7, we performed RNA sequencing on CLCuD‐infested Mac7 and validated RNA‐Seq data with qPCR on 24 independent genes. We performed co‐expression network and pathway analysis for regulation of geminivirus/betasatellite‐interacting genes. We identified nine novel modules with 52 hubs of highly connected genes in network topology within the co‐expression network. Analysis of these hubs indicated the differential regulation of auxin stimulus and cellular localization pathways in response to CLCuD. We also analysed the differential regulation of geminivirus/betasatellite‐interacting genes in Mac7. We further performed the functional validation of selected candidate genes via virus‐induced gene silencing (VIGS). Finally, we evaluated the genomic context of resistance responsive genes and found that these genes are not specific to A or D sub‐genomes of G. hirsutum. These results have important implications in understanding CLCuD resistance mechanism and developing a durable resistance in cultivated cotton.     

Proteomic analysis of stress‐related proteins and metabolic pathways in Picea asperata somatic embryos during partial desiccation

Partial desiccation treatment (PDT) stimulates germination and enhances the conversion of conifer somatic embryos. To better understand the mechanisms underlying the responses of somatic embryos to PDT, we used proteomic and physiological analyses to investigate these responses during PDT in Picea asperata. Comparative proteomic analysis revealed that, during PDT, stress‐related proteins were mainly involved in osmosis, endogenous hormones, antioxidative proteins, molecular chaperones and defence‐related proteins. Compared with those in cotyledonary embryos before PDT, these stress‐related proteins remained at high levels on days 7 (D7) and 14 (D14) of PDT. The proteins that differentially accumulated in the somatic embryos on D7 were mapped to stress and/or stimuli. They may also be involved in the glyoxylate cycle and the chitin metabolic process. The most significant difference in the differentially accumulated proteins occurred in the metabolic pathways of photosynthesis on D14. Furthermore, in accordance with the changes in stress‐related proteins, analyses of changes in water content, abscisic acid, indoleacetic acid and H₂O₂ levels in the embryos indicated that PDT is involved in water‐deficit tolerance and affects endogenous hormones. Our results provide insight into the mechanisms responsible for the transition from morphologically mature to physiologically mature somatic embryos during the PDT process in P. asperata.     

Compensation to simulated insect leaf herbivory in wild cotton (Gossypium hirsutum): responses to multiple levels of damage and associated traits

• Identifying the mechanisms of compensation to insect herbivory remains a major challenge in plant biology and evolutionary ecology. Most previous studies have addressed plant compensatory responses to one or two levels of insect herbivory, and the underlying traits mediating such responses remain elusive in many cases.
• We evaluated responses associated with compensation to multiple intensities of leaf damage (0% control, 10%, 25%, 50%, 75% of leaf area removed) by means of mechanical removal of foliar tissue and application of a caterpillar (Spodoptera exigua) oral secretions in 3‐month‐old wild cotton plants (Gossypium hirsutum). Four weeks post‐treatment, we measured plant growth and multiple traits associated with compensation, namely: changes in above‐ and belowground, biomass and the concentration of nutrients (nitrogen and phosphorus) and non‐structural carbon reserves (starch and soluble sugars) in roots, stems and leaves.
• We found that wild cotton fully compensated in terms of growth and biomass allocation when leaf damage was low (10%), whereas moderate (25%) to high leaf damage in some cases led to under‐compensation. Nonetheless, high levels of leaf removal (50% and 75%) in most cases did not cause further reductions in height and allocation to leaf and stem biomass relative to low and moderate damage. There were significant positive effects of leaf damage on P concentration in leaves and stems, but not roots, as well as a negative effect on soluble sugars in roots.
• These results indicate that wild cotton fully compensated for a low level of leaf damage but under‐compensated under moderate to high leaf damage, but can nonetheless sustain growth despite increasing losses to herbivory. Such responses were possibly mediated by a re‐allocation of carbohydrate reserves from roots to shoots.

     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Stimulation of somatic embryo formation by mutagens and darkness in culture of immature zygotic embryos of Arabidopsis thaliana (L.) Heynh.

The aim of the study was to evaluate the influence of light conditions,physical state of the induction medium and the mutagenic treatment on theembryogenic ability of Arabidopsis thaliana (L.) immaturezygotic embryos differing in developmental stage. The efficiency of directsomatic embryogenesis (DSE) was analysed in a culture of immature zygoticembryos at an early (ES) and a late (LS) developmental stage. The efficiency ofDSE was scored as a percentage of the explants producing somatic embryos. Theexperiments indicated that the physical state of the induction medium (solid orliquid) did not influence the embryogenic ability of the cultured explants. Inthe cultures on both solid and liquid induction medium, the ES explantsproducedsomatic embryos with a frequency of 25.8–37.3% i.e. 2.5–3-timeslower than LS explants. However, an increase in the embryogenic ability of ESexplants (up to 69.8%) was observed when DSE was induced in darkness. Moreover,the stimulation of DSE efficiency in culture of ES explants was also observedafter mutagenic treatment. The chemical mutagens, MNH and EMS, applied forexplant treatment, both stimulated efficiency of somatic embryo formation inculture of ES explants. The most effective DSE induction was observed when MNHand EMS were applied in doses of 0.125–1.0 mM × 3h and0.05–0.2% × 18h, respectively. In these treatment combinations thefrequency of ES explants forming somatic embryos was found to be about 2 timeshigher than in the control culture.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.