Acquisition Of Desiccation Tolerance Research Articles

Embryo Drying Rates during the Acquisition of Desiccation Tolerance in Maize Seed

ABSTRACTMaize (Zea mays L.) seed quality is often reduced because of drying injury, although the causes and impairment mechanisms are poorly understood. In this study, we investigated changes in embryo drying rates and their effect on the acquisition of desiccation tolerance in maize seed. Ears of hybrid maize [B73 × (H99 × H95)] were harvested at about 550, 500, 400, and 320 g H2O kg−1 fresh weight (fw) and subjected to preconditioning (PC) (ear drying at 35°C and 0.47 m s−1 airflow rate) for 0, 12, 24, 36, and 48 h before fluidized bed (FB) drying (shelled seed, 35°C and 5.10 m s−1 airflow rate) treatments to decrease moisture content (MC) to about 130 g H2O kg−1 fw. Additionally, ears were entirely dried under PC (35C) and unheated‐air (NH) conditions. At the four harvests, different drying rate phases were evident in embryos of seed dried entirely at PC (35C) conditions. A slower drying phase coincided with the PC period, which increased with increasing maturation. Under FB drying, embryo MC declined at a faster rate down to about 400 g H2O kg−1 fw, followed by an intermediate drying rate down to about 200 g H2O kg−1 fw, and a slower drying rate below this point. As embryo MC declined to 400 g H2O kg−1 fw at slower drying rates, either with PC or field drying, the ability to withstand the faster drying rates of the FB progressively increased. This effect was illustrated by lower cell solute leakage and better performance in germination and vigor tests. We conclude that slow embryo drying rates to threshold levels may be crucial to acquire their ability to withstand higher drying rates without detrimental effect on seed germination and vigor.

Alignment of Lipid Bodies along the Plasma Membrane during the Acquisition of Desiccation Tolerance in Maize Seed

ABSTRACTThe impairment of lipid body alignment along the plasma membrane during artificial drying of maize (Zea mays L.) seed has been associated with decreases in germination and vigor. In this study, we question further its potential functions as well as the alignment mechanisms. Ears of hybrid maize [B73 × (H99 × H95)] were harvested at about. 550, 500, 400, and 320 g H2O kg−1 fresh weight (fw) and subjected to preconditioning (PC) (ear drying at 35°C and 0.47 m s−1 airflow rate) for 0, 12, 24, 36, and 48 h before fast drying (shelled seed, 35°C and 5.10 m s−1 airflow rate) treatments to decrease seed moisture content (MC) to about 130 g H2O kg−1 fw. In a cross section of the embryo radicle, alignment of lipid bodies (LB) occurred first in the root cap, followed by outer quiescent center (QC) cells and inner QC cells. This alignment pattern was observed during PC and under field drying conditions. Cell plasmolysis and aberrations of the LB were evident in seed harvested at MC > 400 g H2O kg−1 fw and subjected to fast drying without PC. Lipid body aberrations decreased in severity with PC, seed development, and field drying. The alignment of LB coincided with changes in embryo‐drying rates. Decreases in lipid body aberrations coincided with decreases in cell solute leakage and shoot/root ratio and increases in germination percentage. We conclude that alignment of LB along the plasma membrane is a progressive process that occurs from the periphery to the inner QC cells of the embryo radicle. Alignment of LB along the plasma membrane may change cell water relations leading to a more organized dehydration during seed drying. High embryo drying rates may prevent alignment of LB along the plasma membrane and are associated with low germination and vigor.

Isolation and expression of Lea gene in desiccation-tolerant microspore-derived embryos in Brassica spp.

Desiccation tolerance can be induced in microspore-derived embryos of Brassica spp. by application of abscisic acid (ABA). As part of our effort to elucidate the molecular mechanism in induction of desiccation tolerance of embryos, we have isolated Lea genes and examined their expression in microspore-derived embryos of B. napus and B. campestris. The cDNA clones of Lea genes were isolated from desiccation-tolerant microspore-derived embryos of both species. Two cDNA clones of B. napus and B. campestris shared a similar homology of 92.9% identity between the two deduced proteins. These proteins also had high homology with Lea 76 which was isolated from zygotic embryos of B. napus, but were 54 amino acids shorter than Lea 76. Acquisition of desiccation tolerance of the microspore-derived embryos and accumulation of mRNA and its transcripts of the Lea genes began within 12 h of ABA treatment. The embryos treated with ABA for 48-168 h showed a high desiccation tolerance and accumulation of Lea proteins at high levels. The close relationships between the induction of desiccation tolerance of embryos and the expression level of Lea genes suggest an important function of Lea proteins for desiccation tolerance in embryos.

CpR18, a novel SAP-domain plant transcription factor, binds to a promoter region necessary for ABA mediated expression of the CDeT27-45 gene from the resurrection plant Craterostigma plantagineum Hochst.

CDeT27-45 is a lea-like gene from the resurrection plant Craterostigma plantagineum (Scrophulariaceae) which is strongly expressed in vegetative tissues in response to dehydration or treatment with abscisic acid (ABA). Expression of the gene is correlated with the acquisition of desiccation tolerance. Nuclear proteins bind to a 29-bp cis-regulatory region of the promoter which is essential for transcriptional activation of the CDeT27-45 gene by ABA. Using a yeast one-hybrid screen, the cDNA clone CpR18 was isolated, which encodes a protein with specific binding activity for the cis-regulatory element in the CDeT27-45 promoter. The protein contains an acidic region, a SAP-domain, a zinc finger of the C3H-type, and two motifs which are conserved in proteins from several plant species. One of the conserved regions is rich in basic residues and is predicted to form a helix-loop-helix structure. The R18 gene shows high similarities to genomic sequences and ESTs from other plant species. The tissue-specific expression pattern of the rare R18 mRNA and the distribution of nuclear protein binding activity for the CDeT27-45 promoter fragment are compared. The R18 protein is indeed localized in the nucleus, and activates transcription of CDeT27-45 promoter-GUS fusion constructs in tobacco protoplasts. DNA blot analysis and isolation of genomic clones reveal that two copies of R18 are present in the C. plantagineum genome.

In vivo characterization of the effects of abscisic acid and drying protocols associated with the acquisition of desiccation tolerance in alfalfa (Medicago sativa L.) somatic embryos.

Although somatic embryos of alfalfa (Medicago sativa L.) had acquired some tolerance to desiccation at the cotyledonary stage of development (22 d after plating), additional culturing in 20 microm abscisic acid (ABA) for 8 d induced greater desiccation tolerance, as determined by increased germination. Compared with fast drying, slow drying of the ABA-treated embryos improved desiccation tolerance. However, slow drying of non-ABA-treated embryos led to the complete loss of germination capacity, while some fast-dried embryos survived. An electron paramagnetic resonance spin probe technique and in vivo Fourier transform infrared microspectroscopy revealed that cellular membrane integrity and a-helical protein secondary structure were maintained during drying in embryos cultured in media enriched with 20 microM ABA, but not in embryos cultured in the absence of ABA. Slow-dried, non-ABA-treated embryos had low oligosaccharide to sucrose ratios, an increased proportion of beta-sheet protein secondary structures and broad membrane phase transitions extending over a temperature range of more than 60 degrees C, suggestive of irreversible phase separations. The spin probe study showed evidence of imbibitional damage, which could be alleviated by prehydration in humid air. These observations emphasize the importance of appropriate drying and prehydration protocols for the survival and storage of somatic embryos. It is suggested that ABA also plays a role in suppressing metabolism, thus increasing the level of desiccation tolerance; this is particularly evident under stressful conditions such as slow drying.

An integrated overview of seed development in Arabidopsis thaliana ecotype WS

This work is part of a research program aiming at identifying and studying genes involved in Arabidopsis thaliana seed maturation. We focused here on the Wassilewskija ecotype seed development and linked physiological and biochemical data, including protein, oil, soluble sugars, starch and free amino acid measurements, to embryo development, to obtain a complete and thorough reference data set. A. thaliana seed development can be divided into three stages. During early embryogenesis (i.e. morphogenesis), seed weight and lipid content were low whereas important amounts of starch were transiently accumulated. In the second stage, or maturation phase, a rapid increase in seed dry weight was observed and storage oils and proteins were accumulated in large quantities, accounting for approximately 40% of dry matter each at the end of this stage. During the third and last stage (late maturation including acquisition of desiccation tolerance), seed dry weight remained constant while an acute loss of water took place in the seed. Storage compound synthesis ended concomitantly with sucrose, stachyose and raffinose accumulation. This study revealed the occurrence of metabolic activities such as protein synthesis, in the final phase of embryo desiccation. A striking correlation between peaks in hexose to sucrose ratio and transition phases during embryogenesis was observed.

Abscisic acid and desiccation tolerance in oil palm (Elaeis guineensis Jacq.) somatic embryos

The effect of exogenous abscisic acid (ABA) on the desiccation tolerance, soluble sugar contents and germination rates of oil palm somatic embryos was studied. ABA was added during the development or germination of the somatic embryos. The treatments had no effect on embryo dry weights or water and sucrose contents. In contrast, monosaccharide levels decreased in ABA-treated embryos and raffinose was detected in embryos treated for 6 weeks with increasing concentrations of ABA (from 5 to 25 μM). ABA significantly improved embryo tolerance to rapid desiccation. Embryos treated with 25 μM ABA exhibited survival rates higher than that of the control. The 2-week ABA treatment induced a delay in shoot emission during embryo germination on hormone-free media. In contrast, embryos treated with 50 μM ABA displayed no germination. The presence of ABA in germination media induced inhibition of embryo shoot emission. These results suggest that ABA plays a role in the maturation and the acquisition of desiccation tolerance of oil palm somatic embryos.

The competence to acquire cellular desiccation tolerance is independent of seed morphological development.

Acquisition of desiccation tolerance and the related changes at the cellular level in wheat (Triticum aestivum cv. Priokskaya) kernels during normal development and premature drying on the ear were studied using a spin probe technique and low temperature scanning electron microscopy. During normal development, the ability of embryos to germinate after rapid drying and rehydration was acquired after completion of morphological development, which is a few days before mass maturity. The acquisition of desiccation tolerance, as assessed by germination, was associated with an upsurge in cytoplasmic viscosity, the onset of accumulation of protein and oil bodies, and the retention of membrane integrity upon dehydration/rehydration. These features were also used to assess cellular desiccation tolerance in the cases when germination could not occur. Slow premature drying was used to decouple the acquisition of cellular desiccation tolerance from morphogenesis. Upon premature drying of kernels on the ears of plants cut at 5 d after anthesis, desiccation-tolerant dwarf embryos were formed that were able to germinate. When plants were cut at earlier stages poorly developed embryos were formed that were unable to germinate, but cellular desiccation tolerance was nevertheless acquired. In such prematurely dried kernels, peripheral meristematic endosperm cells had already passed through similar physiological and ultrastructural changes associated with the acquisition of cellular desiccation tolerance. It is concluded that despite the apparent strong integration in seed development, desiccation tolerance can be acquired by the meristematic cells in the developing embryo and cambial layer of endosperm, independently of morphological development.

Changes in leaf hexokinase activity and metabolite levels in response to drying in the desiccation-tolerant species Sporobolus stapfianus and Xerophyta viscosa.

The phosphorylation of glucose and fructose is an important step in regulating the supply of hexose sugars for biosynthesis and metabolism. Changes in leaf hexokinase (EC 2.7.1.1) activity and in vivo metabolite levels were examined during drying in desiccation-tolerant Sporobolus stapfianus and Xerophyta viscosa. Leaf hexokinase activity was significantly induced from 85% to 29% relative water content (RWC) in S. stapfianus and from 89% to 55% RWC in X. viscosa. The increase in hexokinase corresponded to the region of sucrose accumulation in both species, with the highest activity levels coinciding with region of net glucose and fructose removal. The decline of hexose sugars and accumulation of sucrose in both plant species was not associated with a decline in acid and neutral invertase. The increase in hexokinase activity may be important to ensure that the phosphorylation and incorporation of glucose and fructose into metabolism exceeded production from potential hydrolytic activity. Total cellular glucose-6-phosphate (Glc-6-P) and fructose-6-phosphate (Fru-6-P) levels were held constant throughout dehydration. In contrast to hexokinase, fructokinase activity was unchanged during dehydration. Hexokinase activity was not fully induced in leaves of S. stapfianus dried detached from the plant, suggesting that the increase in hexokinase may be associated with the acquisition of desiccation-tolerance.

Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis.

Seed oligosaccharides (OSs) and especially raffinose series OSs (RSOs) are hypothesized to play an important role in the acquisition of desiccation tolerance and consequently in seed storability. In the present work we analyzed the seed-soluble OS (sucrose, raffinose, and stachyose) content of several Arabidopsis accessions and thus identified the genotype Cape Verde Islands having a very low RSO content. By performing quantitative trait loci (QTL) mapping in a recombinant inbred line population, we found one major QTL responsible for the practically monogenic segregation of seed stachyose content. This locus also affected the content of the two other OSs, sucrose, and raffinose. Two candidate genes encoding respectively for galactinol synthase and raffinose synthase were located within the genomic region around this major QTL. In addition, three smaller-effect QTL were identified, each one specifically affecting the content of an individual OS. Seed storability was analyzed in the same recombinant inbred line population by measuring viability (germination) under two different seed aging assays: after natural aging during 4 years of dry storage at room temperature and after artificial aging induced by a controlled deterioration test. Thus, four QTL responsible for the variation of this trait were mapped. Comparison of the QTL genetic positions showed that the genomic region containing the major OS locus did not significantly affect the seed storability. We concluded that in the studied material neither RSOs nor sucrose content had a specific effect on seed storability.

Effects of dehydration conditions on desiccation tolerance of developing pea seeds as related to oligosaccharide content and cell membrane properties

Germination and carbohydrate metabolism were studied in fresh developing pea (Pisum sativum L., cv Baccara) seeds and after artificial drying at 25°C and various relative humidities (20, 75 and 99% RH) to investigate whether the occurrence of desiccation tolerance was related to sucrose, raffinose and stachyose contents. Seeds became completely tolerant to fast drying at 25°C and 20% RH a few days after the end of reserve accumulation, i.e. when their moisture content dropped to approx. 50% (fresh weight basis). This acquisition of desiccation tolerance was associated with an accumulation of raffinose and stachyose, the latter being more abundant in the embryonic axis than in the cotyledons. The (raffinose+stachyose)/sucrose ratio increased during seed development and reached 1.1 in the axis and 0.2 in the cotyledons just before the onset of desiccation tolerance. When the natural acquisition of desiccation tolerance occurred on the mother plant, artificial drying of isolated seeds induced an increase in oligosaccharide content in the cotyledons. Immature seeds, the moisture content of which was higher than about 60% (fresh weight basis), did not tolerate fast drying (25°C and 20 or 75% RH). Such drying did not result in the synthesis of stachyose and induced an increase in electrolyte leakage, a decrease in the ability of seeds to convert 1-aminocyclopropane 1-carboxylic acid (ACC) to ethylene and an increase in ethane synthesis, thus indicating a deterioration of cell membrane properties and lipid peroxidation. In contrast, immature seeds tolerated drying either in the pods or at 25°C and 99% RH, and such drying induced a decrease in sucrose content, an increase in oligosaccharide content and a (raffinose+stachyose)/sucrose ratio higher than around 1. Soluble sugar contents of dried immature seeds depended on the conditions of dehydration. In cotyledons, the (raffinose+stachyose)/sucrose ratio reached 0.61 when seeds were dried at 25°C and 99% RH, whereas it was as low as 0.15 when drying was performed at 25°C and 20% RH. All the results obtained are consistent with the concept that oligosaccharides may well be involved in the protection of membranes during dehydration.

Acquisition of Desiccation Tolerance by Cultured Carrot Cells upon Ectopic Expression of C-ABI3, a Carrot Homolog of ABI3

To obtain direct evidence for the involvement of C-ABI3, a carrot ( Daucus carota L.) homolog of VP I/ABI3, seed-specific transcription factor, in the acquisition of desiccation tolerance, we prepared transgenic carrot non-embryogenic cells (NC) in which the C-ABI3 gene was expressed ectopically. Non-transgenic NC, in which expression of C-ABI3 was not detected, did not exhibit desiccation tolerance even after treatment with abscisic acid (ABA). In transgenic NC that expressed C-ABI3 , embryo-specific ABA-inducible genes ( ECP genes) were expressed upon ABA-treatment. Furthermore, the transgenic NC became desiccation-tolerant upon ABA-treatment, bur did not tolerate desiccation without ABA-treatment.

Changed properties of the cytoplasmic matrix associated with desiccation tolerance of dried carrot somatic embryos. An In situ fourier transform infrared spectroscopic study

Abscisic acid-pretreated carrot (Daucus carota) somatic embryos survive dehydration upon slow drying, but fast drying leads to poor survival of the embryos. To determine whether the acquisition of desiccation tolerance is associated with changes in the physical stability of the cytoplasm, in situ Fourier transform infrared microspectroscopy was used. Although protein denaturation temperatures were similar in the embryos after slow or fast drying, the extent of the denaturation was greater after fast drying. Slowly dried embryos are in a glassy state at room temperature, and no clearly defined glassy matrix was observed in the rapidly dried embryos. At room temperature the average strength of hydrogen bonding was much weaker in the rapidly dried than in the slowly dried embryos. We interpreted the molecular packing to be "less tight" in the rapidly dried embryos. Whereas sucrose (Suc) is the major soluble carbohydrate after fast drying, upon slow drying the trisaccharide umbelliferose accumulates at the expense of Suc. The possibly protective role of umbelliferose was tested on protein and phospholipid model systems, using Suc as a reference. Both umbelliferose and Suc form a stable glass with drying: They depress the transition temperature of dry liposomal membranes equally well, they both prevent leakage from dry liposomes after rehydration, and they protect a polypeptide that is desiccation sensitive. The similar protection properties in model systems and the apparent interchangeability of both sugars in viable, dry somatic embryos suggest no special role of umbelliferose in the improved physical stability of the slowly dried embryos. Also, during slow drying LEA (late-embryogenesis abundant) transcripts are expressed. We suggest that LEA proteins embedded in the glassy matrix confer stability to these slowly dried embryos.

Analyses to determine the role of embryo immaturity in dormancy maintenance of yellow cedar (Chamaecyparis nootkatensis) seeds: synthesis and accumulation of storage proteins and proteins implicated in desiccation tolerance

Development of yellow cedar seeds is completed by about 17-21 months after pollination. Following dispersal from the parent plant, the seeds exhibit a low capacity for germination and typically require an additional year to meet their moist chilling requirements and break dormancy. Biochemical analyses were undertaken in order to address whether seed dormancy is imposed and maintained because the embryo or megagametophyte is immature at the time of seed shedding and hence requires time to complete developmental events before dormancy can be terminated. Major protein reserves of the embryo and megagametophyte are the buffer-insoluble crystalloid (legumin) storage proteins and the water-soluble albumin proteins. SDS-PAGE, fluorography of in vivo synthesized proteins and Western blot analyses showed that the greatest increase in protein reserve synthesis and accumulation occurred between the first and second years of development; deposition of soluble and insoluble storage protein was largely completed in seeds of second-year cones by August, 2-3 months prior to seed dispersal. The period associated with greatest accumulation of storage proteins was accompanied by an increased accumulation of two ER-resident proteins associated with post-translational maturation of storage proteins (binding protein and protein disulphide isomerase). Accumulation of proteins implicated in the acquisition of desiccation tolerance (dehydrins and the tonoplast intrinsic protein, α-TiP) occurred between the first and second years of development. Several heat-stable proteins and some of the proteins associated with late development continued to be synthesized after seed shedding and in 13 d moist-chilled mature seeds. However, this did not include the major dehydrin-like protein of yellow cedar seeds. Further, the continued synthesis of heat-stable proteins does not appear to be a factor preventing the germination of yellow cedar seeds following dispersal from the parent plant; rather, the mechanism of dormancy is primarily coat-imposed.

Irrigation and Seed Quality Development in Rapid-cycling Brassica: Soluble Carbohydrates and Heat-stable Proteins

Abstract Changes in soluble carbohydrates and heat-stable proteins have been examined in relation to the acquisition of desiccation tolerance and/or potential seed longevity during seed development in rapid-cycling brassica [ Brassica campestris (rapa) L.]. Rates of seed development were moderated by different irrigation regimes. At the early stages, glucose, fructose and sucrose predominated. The raffinose series oligosaccharides accumulated during seed maturation, and occurred earliest in seeds from plants irrigated only until 16 days after pollination. Stachyose content correlated positively, and monosaccharide content correlated negatively, with the ability of seeds to tolerate rapid desiccation and with their potential longevity (the constant K i of the seed viability equation). Similarly, the ratio of oligosaccharide:total sugars provided strong positive correlations with ability to tolerate desiccation and with potential longevity. Most of the heat-stable proteins selected for study accumulated comparatively late, i.e. during maturation drying. The imposition of water stress induced earlier accumulation of heat-stable proteins. The ability to tolerate desiccation was correlated with the content of selected heat-stable proteins, but potential longevity provided stronger correlations. The content of a 58 kDa heat-stable protein provided the strongest positive correlation with potential longevity. A simple multiple regression model of the relations between potential longevity and both the oligosaccharide:total sugar ratio and the 58 kDa heat-stable protein content was developed for all three plant irrigation regimes to show the combined effect of certain sugars and proteins on seed quality. The model suggests that these sugars and proteins are equally likely to be required for seed quality development, and that initially the sugars tend to accumulate at a greater rate than the proteins, but that during maturation drying the heat-stable proteins accumulate at the greater rate.

Fagopyritol B1, O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol, a galactosyl cyclitol in maturing buckwheat seeds associated with desiccation tolerance.

O-alpha-D-Galactopyranosyl-(1-->2)-D-chiro-inositol, herein named fagopyritol B1, was identified as a major soluble carbohydrate (40% of total) in buckwheat (Fagopyrum esculentum Moench, Polygonaceae) embryos. Analysis of hydrolysis products of purified compounds and of the crude extract led to the conclusion that buckwheat embryos have five alpha-galactosyl D-chiro-inositols: fagopyritol A1 and fagopyritol B1 (mono-galactosyl D-chiro-inositol isomers), fagopyritol A2 and fagopyritol B2 (di-galactosyl D-chiro-inositol isomers), and fagopyritol B3 (tri-galactosyl D-chiro-inositol). Other soluble carbohydrates analyzed by high-resolution gas chromatography included sucrose (42% of total), D-chiro-inositol, myo-inositol, galactinol, raffinose and stachyose (1% of total), but no reducing sugars. All fagopyritols were readily hydrolyzed by alpha-galactosidase (EC 3.2.1.22) from green coffee bean, demonstrating alpha-galactosyl linkage. Retention time of fagopyritol B1 was identical to the retention time of O-alpha-D-galactopyranosyl-(1-->2)-D-chiro-inositol from soybean (Glycine max (L.) Merrill, Leguminosae), suggesting that the alpha-galactosyl linkage is to the 2-position of D-chiro-inositol. Accumulation of fagopyritol B1 was associated with acquisition of desiccation tolerance during seed development and maturation in planta, and loss of fagopyritol B1 correlated with loss of desiccation tolerance during germination. Embryos of seeds grown at 18 degrees C, a condition that favors enhanced seed vigor and storability, had a sucrose-to-fagopyritol B1 ratio of 0.8 compared to a ratio of 2.46 for seeds grown at 25 degrees C. We propose that fagopyritol B1 facilitates desiccation tolerance and storability of buckwheat seeds.

Nuclear Replication Activity During Seed Development, Dormancy Breakage and Germination in Three Tree Species: Norway Maple (Acer platanoidesL.), Sycamore (Acer pseudoplatanusL.) and Cherry (Prunus aviumL.)

Flow cytometric analyses of nuclear DNA levels were carried out during development, stratification and germination of dormant seeds from three tree species with contrasting characteristics. Norway maple (Acer platanoides) and sycamore (Acer pseudoplatanus) have orthodox (desiccation-tolerant) and recalcitrant (desiccation-sensitive) storage behaviours, respectively, and require only a period of cold to break dormancy, whereas, orthodox cherry (Prunus avium) seeds require an initial warm period before cold stratification to fully stimulate germination. Whole embryos and radicle tips of both Norway maple and sycamore were found to have stable high levels of 4C DNA during the latter stages of development and both contained nuclei arrested at the 2C and 4C levels at maturity. Mature cherry embryos had nuclei predominantly arrested at the 2C level. This suggests that the acquisition of desiccation tolerance is not correlated with the arrest of the cell cycle at any particular nuclear DNA level. Neither DNA replication in radicle cells nor germination occurred when seeds were maintained moist at a constant 20 °C. However, in the late stages of cold treatment during stratification, nuclear DNA levels in radicle cells changed in advance of radicle emergence in the orthodox Norway maple and cherry, whereas in the recalcitrant sycamore, change was not recorded until after radicle emergence. These results show that DNA replication has potential use as an indicator of the progress of tree seeds through stratification treatments used to break some types of dormancy. The ways in which this indicator could be exploited for seed quality and performance testing are discussed.

Fourier transform infrared microspectroscopy detects changes in protein secondary structure associated with desiccation tolerance in developing maize embryos

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of alpha-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the alpha-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of alpha-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.

Raffinose accumulation in maize embryos in the absence of a fully functional Vp1 gene product

The maize (Zea mays L.) Viviparous-1 (Vp1) gene encodes the cotranscriptional activator VP1 protein and regulates biosynthesis of anthocyanins, sensitivity to abscisic acid, repression of α-amylase, and other factors related to quiescence during embryo maturation. Five mutant vp1 alleles were used to determine if the Vp1 gene regulates raffinose accumulation and the onset of desiccation tolerance in isolated embryos. Embryos of wild-type Vp1-R (purple, non-viviparous) kernels acquired desiccation tolerance to fast drying in association with a sucrose:raffinose mass ratio of 10:1 or lower. After slow drying, germinability was related to stage of embryo development. Embryos of mutant vp1-R (yellow, viviparous) kernels, in which the Vp1 gene is not expressed, accumulated only trace amounts of raffinose and never acquired desiccation tolerance. Embryos from kernels (yellow, non-viviparous) carrying the modified mutant allele vp1-McWhirter (which encodes a VP1 protein that is truncated at the C-terminal end), the “Coe” mutant allele vp1-1695, or the mutant alleles vp1-A1 or vp1-C821708 all become quiescent. Developing embryos of kernels with the modified mutant alleles vp1-McWhirter and vp1-1695 were desiccation tolerant in association with the sucrose:raffinose mass ratio having a value of 20:1 or lower. Embryos from wild-type kernels segregated on the same ears (Vp1-1695, purple) also were desiccation tolerant in association with a sucrose:raffinose mass ratio of less than 20:1. The acquisition of desiccation tolerance in developing maize embryos may be associated with raffinose accumulation, and raffinose biosynthesis may occur in the absence of a fully functional Vp1 gene product.

Oligosaccharides and galactosyl cyclitols in seed desiccation tolerance

AbstractSoluble carbohydrates are one of multiple components required for the acquisition of desiccation tolerance during seed development and maturation. Sucrose and the raffinose series of oligosaccharides have been extensively studied in relation to seed desiccation tolerance. These galactosyl sucrose oligosaccharides are present in viable tissues of many edible seeds, especially the legumes, and contribute to flatulence after ingestion. A reduction in oligosaccharides of the raffinose series is desired by nutritionists but, if present at less than a threshold level, this this may result in reduced desiccation tolerance and storability of seeds. Some seeds that have very low amounts of raffinose and stachyose accumulate galactosyl cyclitols and small amounts of free cyclitols. Galactosyl cyclitols present in various seeds include galactosyl or methyl derivatives ofmyo-inositol, D-pinitol, D-chiro-inositol, D-ononitol, andscyllo-inositol. Castor bean seeds accumulate galactinol and buck-wheat seeds accumulate fagopyritol in addition to sucrose. Fagopyritol accumulation is associated with the acquisition of desiccation tolerance in buckwheat seeds. The galactosyl sucrose and galactosyl cyclitol soluble carbohydrates in maturing seeds have been proposed to have various roles in desiccation tolerance including non-toxic and non-reducing forms of seed storage products and intracellular osmotic agents contributing to the structural stability of organelles, membranes, enzymes and proteins, other macromolecules, and the glassy state. Ether derivatives of cyclitols may form liquid crystals. Methyl ether derivatives of cyclitols may have roles as cryoprotectants, desiccation protectants, and hydroxyl radical scavengers.