Kernel Abortion in Maize : II. Distribution of C among Kernel Carbohydrates

This study was designed to compare the uptake and distribution of ¹⁴C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 30 and 35°C were transferred to [¹⁴C]sucrose media 10 days after pollination. Kernels cultured at 35°C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected after 24 hours in culture on labeled media. After 8 days in culture on [¹⁴C]sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35°C, respectively. This indicates that some of the sucrose taken up by the cob tissue was cleaved to fructose and glucose in the cob. Of the total carbohydrates, a higher percentage of label was associated with sucrose and a lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35°C compared to kernels cultured at 30°C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35°C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30°C (89%). Kernels cultured at 35°C had a correspondingly higher proportion of ¹⁴C in endosperm fructose, glucose, and sucrose. These results indicate that starch synthesis in the endosperm is strongly inhibited in kernels induced to abort by high temperature even though there is an adequate supply of sugar.     

Effect of increased temperature in apical regions of maize ears on starch-synthesis enzymes and accumulation of sugars and starch

Apical florets of maize (Zea mays L.) ears differentiate later than basal florets and form kernels which have lower dry matter accumulation rates. The purpose of this study was to determine whether increasing the temperature of apical kernels during the dry matter accumulation period would alter the difference in growth rate between apical and basal kernels. Apical regions of field-grown maize (cultivar Cornell 175) ears were heated to 25 ± 3°C from 7 days after pollination to maturity (tip-heated ears) and compared with unheated ears (control). In controls, apical-kernel endosperm had 24% smaller dry weight at maturity, lower concentration of sucrose, and lower activity of ADP-Glc starch synthase than basal-kernel endosperm, whereas ADP-Glc-pyrophosphorylase (ADPG-PPase) activities were similar. In tip-heated ears apical-kernel endosperm had the same growth rate and final weight as basal-kernel endosperm and apical kernels had higher sucrose concentrations, higher ADP-Glc starch synthase activity, and similar ADPG-PPase activity. Total grain weight per ear was not increased by tip-heating because the increase in size of apical kernels was partially offset by a slight decrease in size of the basal- and middle-position kernels. Tip-heating hastened some of the developmental events in apical kernels. ADPG-PPase and ADP-Glc starch synthase activities reached peak levels and starch concentration began rising earlier in apical kernels. However, tip-heating did not shorten the period of starch accumulation in apical kernels. The results indicate that the lower growth rate and smaller size of apical kernels are not solely determined by differences in prepollination floret development.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.     

Growth and composition of maize kernels cultured in vitro with varying supplies of carbon and nitrogen

This study employed in vitro seed culture to determine how C and N supply influence the growth (i.e. starch accumulation) and protein composition of maize (Zea mays L.) endosperm. Immature kernels were grown to maturity on liquid medium containing various concentrations of C (sucrose at 234 millimolar [low] and 468 millimolar [high]) and N (amino acid mixture ranging in N from 0 to 144 millimolar). Low C supply limited starch, but not N, accumulation in the endosperm. With high C, endosperm starch and protein content increased concomitantly as N supply increased from 0 to 13.4 millimolar. Endosperm growth was unaffected by additional N until concentrations exceeding approximately 72 millimolar reduced starch accumulation. A similar inhibition of starch deposition occurred with lower N concentrations when kernels were grown with low C. Endosperm total N content reached a point of saturation with approximately 36 millimolar N in the medium, regardless of C supply. Zein synthesis in the endosperm responded positively across all N levels, while glutelin content remained static and albumin/globulin proteins were reduced in amount when N supply was greater than 36 millimolar. A reciprocal, inverse relationship was observed in mature endosperm tissue between the concentrations of free amino acids and soluble sugars. Our data suggest that under N stress starch and protein accumulation in the endosperm are interdependent, at least in appearance, but are independent otherwise.     

Brittle-1, an Adenylate Translocator, Facilitates Transfer of Extraplastidial Synthesized ADP-Glucose into Amyloplasts of Maize Endosperms

Amyloplasts of starchy tissues such as those of maize (Zea mays L.) function in the synthesis and accumulation of starch during kernel development. ADP-glucose pyrophosphorylase (AGPase) is known to be located in chloroplasts, and for many years it was generally accepted that AGPase was also localized in amyloplasts of starchy tissues. Recent aqueous fractionation of young maize endosperm led to the conclusion that 95% of the cellular AGPase was extraplastidial, but immunolocalization studies at the electron- and light-microscopic levels supported the conclusion that maize endosperm AGPase was localized in the amyloplasts. We report the results of two nonaqueous procedures that provide evidence that in maize endosperms in the linear phase of starch accumulation, 90% or more of the cellular AGPase is extraplastidial. We also provide evidence that the brittle-1 protein (BT1), an adenylate translocator with a KTGGL motif common to the ADP-glucose-binding site of starch synthases and bacterial glycogen synthases, functions in the transfer of ADP-glucose into the amyloplast stroma. The importance of the BT1 translocator in starch accumulation in maize endosperms is demonstrated by the severely reduced starch content in bt1 mutant kernels.     

Pollen source effects on growth of kernel structures and embryo chemical compounds in maize

Background and Aims

Previous studies have reported effects of pollen source on the oil concentration of maize (Zea mays) kernels through modifications to both the embryo/kernel ratio and embryo oil concentration. The present study expands upon previous analyses by addressing pollen source effects on the growth of kernel structures (i.e. pericarp, endosperm and embryo), allocation of embryo chemical constituents (i.e. oil, protein, starch and soluble sugars), and the anatomy and histology of the embryos.

Methods

Maize kernels with different oil concentration were obtained from pollinations with two parental genotypes of contrasting oil concentration. The dynamics of the growth of kernel structures and allocation of embryo chemical constituents were analysed during the post-flowering period. Mature kernels were dissected to study the anatomy (embryonic axis and scutellum) and histology [cell number and cell size of the scutellums, presence of sub-cellular structures in scutellum tissue (starch granules, oil and protein bodies)] of the embryos.

Key Results

Plants of all crosses exhibited a similar kernel number and kernel weight. Pollen source modified neither the growth period of kernel structures, nor pericarp growth rate. By contrast, pollen source determined a trade-off between embryo and endosperm growth rates, which impacted on the embryo/kernel ratio of mature kernels. Modifications to the embryo size were mediated by scutellum cell number. Pollen source also affected (P < 0·01) allocation of embryo chemical compounds. Negative correlations among embryo oil concentration and those of starch (r = 0·98, P < 0·01) and soluble sugars (r = 0·95, P < 0·05) were found. Coincidently, embryos with low oil concentration had an increased (P < 0·05–0·10) scutellum cell area occupied by starch granules and fewer oil bodies.

Conclusions

The effects of pollen source on both embryo/kernel ratio and allocation of embryo chemicals seems to be related to the early established sink strength (i.e. sink size and sink activity) of the embryos.Key words: Zea mays, maize, pollen, kernel, embryo, endosperm, oil, protein, starch, soluble sugars     

Endosperm cell division in maize kernels cultured at three levels of water potential

The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of −1.1, −1.6, or −2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of −1.6 or −2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control (−1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the −2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at −1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two −2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors.     

Studies of Frost Hardiness in Woody Plants. II. Effect of Temperature on Hardening

The effect of temperature on hardening was studied at temperatures ranging from 0° to −20° using twigs of willow and poplar. In October and in late April when the twigs are not very frost hardy, hardening at 0° produced a considerable increase in their frost hardiness, although the effectiveness of hardening at 0° decreased with a decrease in the environmental temperature. In twigs which could withstand continuous freezing without injury, hardening at −3° to −5° was most effective in increasing the frost hardiness of the twigs. Below −20°, only negligible increase was observed either in frost hardiness or sugar content.

The rate of starch to sugar conversion differed remarkably in different twig tissues. The starch in xylem was more slowly converted to sugar than that in the cortex. The optimum temperature for converting starch into sugar during frost hardening was also found to be −3° to −5°. In addition, the greater the effectiveness of the hardening treatment, the greater the rate of conversion from starch to sugar. The frost hardiness of a twig is closely related to the sugar content of the twig, especially in the xylem.

     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Rice Chalky Ring Formation Caused by Temporal Reduction in Starch Biosynthesis during Osmotic Adjustment under Foehn-Induced Dry Wind

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s⁻¹) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.     

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/−/−; +/+/−; +/+/+) or incapable (−/−/−) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.     

rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage-dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer-specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 cM away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild-type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild-type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5-10 DAP determines the rgf1 kernel phenotype.     

Nucleoside Diphosphate Sugar-Starch Glucosyl Transferase Activity of wx Starch Granules

Starch granule preparations from the endosperm tissue of all waxy maize (Zea mays L.) mutants tested have low and approximately equal capability to incorporate glucose from adenosine diphosphate glucose into starch. As the substrate concentration is reduced, however, the activity of waxy preparations relative to nonmutant increases until, at the lowest substrate concentration utilized (0.1 μM), the activity of the waxy preparations is nearly equal to that of the nonmutant preparation. The apparent K_m (adenosine diphosphate glucose) for starch granule preparations from wx-C/wx-C/wx-C endosperms was 7.1 × 10⁻⁵ M, which is compared to 3 × 10⁻³ M for preparations from nonwaxy endosperms. Starch granule preparations from three other waxy mutants of independent mutational origin have levels of enzymic activity approximately equal to wx-C at a given substrate concentration giving rise to similar apparent K_m estimates. We conclude that there is in maize endosperm starch granules a second starch granule-bound glycosyl transferase, whose presence is revealed when mutation eliminates activity of the more active glucosyl transferase catalyzing the same reaction.     

Sucrose synthase activity in developing wheat endosperms differing in maximum weight

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the “rate limiting” enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.     

Comparative Proteomic Analyses Provide New Insights into Low Phosphorus Stress Responses in Maize Leaves

Phosphorus deficiency limits plant growth and development. To better understand the mechanisms behind how maize responds to phosphate stress, we compared the proteome analysis results of two groups of maize leaves that were treated separately with 1,000 µM (control, +P) and 5 µM of KH₂PO₄ (intervention group, −P) for 25 days. In total, 1,342 protein spots were detected on 2-DE maps and 15.43% had changed (P<0.05; ≥1.5-fold) significantly in quantity between the +P and −P groups. These proteins are involved in several major metabolic pathways, including photosynthesis, carbohydrate metabolism, energy metabolism, secondary metabolism, signal transduction, protein synthesis, cell rescue and cell defense and virulence. The results showed that the reduction in photosynthesis under low phosphorus treatment was due to the down-regulation of the proteins involved in CO₂ enrichment, the Calvin cycle and the electron transport system. Electron transport and photosynthesis restrictions resulted in a large accumulation of peroxides. Maize has developed many different reactive oxygen species (ROS) scavenging mechanisms to cope with low phosphorus stress, including up-regulating its antioxidant content and antioxidase activity. After being subjected to phosphorus stress over a long period, maize may increase its internal phosphorus utilization efficiency by altering photorespiration, starch synthesis and lipid composition. These results provide important information about how maize responds to low phosphorus stress.     

Sugar Uptake and Metabolism in the Developing Endosperm of Tassel-seed Tunicate (Ts-5 Tu) Maize

Factors regulating assimilate transport into developing maize (Zea mays L.) kernels have been difficult to determine because of the structural complexity of basal kernel tissues and the damage that results from tissue dissection. The sensitivity of maize kernels to experimental manipulation is such that substantial maternal tissue is required to support kernel growth in vitro. Consequently, sugar transport experiments with isolated seed tissues or detached kernels have not unequivocally demonstrated how sugar transport occurs. In the present study, Tassel-seed Tunicate (Ts-5 Tu) maize kernels were investigated as a model system for introducing test solutions into the pedicel apoplast with minimal wounding. Transpiration in leafy glumes drew ¹⁴C-sugar solutions up the 8- to 10-millimeter-long pedicel stalks into the basal endosperm transfer cell region. ¹⁴C from fructose was incorporated into starch for 8 days. Sugar uptake into endosperm and embryo tissue showed specificity and inhibitor sensitivity. In particular, p-chloromercuribenzene sulfonate partially inhibited fructose uptake into the endosperm but had no effect on the metabolic conversion of that fructose that entered the endosperm. These results are consistent with active, carrier-mediated sugar transport, but a definitive determination would require more detailed tissue analysis. We propose that further refinement of the incubation solution may allow long-term kernel growth without cob tissue and thus provide a more precise determination of which maternal factors influence seed development.     

Adaptation of Grapevine Flowers to Cold Involves Different Mechanisms Depending on Stress Intensity

Grapevine flower development and fruit set are influenced by cold nights in the vineyard. To investigate the impact of cold stress on carbon metabolism in the inflorescence, we exposed the inflorescences of fruiting cuttings to chilling and freezing temperatures overnight and measured fluctuations in photosynthesis and sugar content. Whatever the temperature, after the stress treatment photosynthesis was modified in the inflorescence, but the nature of the alteration depended on the intensity of the cold stress. At 4°C, photosynthesis in the inflorescence was impaired through non-stomatal limitations, whereas at 0°C it was affected through stomatal limitations. A freezing night (−3°C) severely deregulated photosynthesis in the inflorescence, acting primarily on photosystem II. Cold nights also induced accumulation of sugars. Soluble carbohydrates increased in inflorescences exposed to −3°C, 0°C and 4°C, but starch accumulated only in inflorescences of plants treated at 0 and −3°C. These results suggest that inflorescences are able to cope with cold temperatures by adapting their carbohydrate metabolism using mechanisms that are differentially induced according to stress intensity.