Near-isogenic lines for measuring phenotypic effects of DIMBOA-Glc methyltransferase activity in maize

Three O-methyltransferases (BX10a, b, c) catalyze the conversion of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) to 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) in maize (Zea mays). Variation in benzoxazinoid accumulation and resistance to Rhopalosiphum maidis (corn leaf aphid) was attributed to a natural CACTA family transposon insertion that inactivates Bx10c. Whereas maize inbred line B73 has this transposon insertion, line CML277 does not. To characterize the phenotypic effects of DIMBOA-Glc methyltransferase activity, we created near-isogenic lines derived from B73 and CML277 that do or do not contain the transposon insertion. Bx10c inactivation causes high DIMBOA-Glc, low HDMBOA-Glc, and decreased aphid reproduction relative to near-isogenic lines that have a functional Bx10c gene. These results confirm the importance of this locus in maize aphid resistance. The availability of Bx10c near-isogenic lines will facilitate further research on the function of different benzoxazinoids and DIMBOA-Glc methyltransferase activity in maize defense against herbivores and pathogens.     

Development of two isogenic sweet corn hybrids differing for glycinebetaine content

A hybrid of sweet corn, Zea mays L. (`1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid `1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize.     

Development and characterization of near-isogenic lines of sorghum segregating for glycinebetaine accumulation

Glycinebetaine (GB) is a compatible solute synthesized by species in a number of plant families, including some grasses such as sorghum ( Sorghum bicolor L. Moench). A recombinant-inbred (RI) population was developed from a cross between GB-deficient (IS2319) and GB-accumulating (P932296) sorghum genotypes. The proportions of GB-accumulating lines in both the F₅ and F_7:8 generations of this RI population were higher than expected, possibly due to some beneficial effect of GB accumulation on plant growth, survival or seed set. GB levels varied widely in lines of the RI population that were grown under controlled conditions, suggesting genetic control not only for the presence or absence of GB, but also for the level of GB. This hypothesis was tested by analysing individual plants from lines identified as accumulating low, medium or high concentrations of GB based on the F₅ and F_7:8 screens. The level of GB was conserved within lines, supporting the hypothesis of genetic control of relative GB levels within accumulating lines. Two pairs of near-isogenic lines (NILs) with contrasting GB levels within pairs were developed from the RI population. The stable inheritance of the GB phenotype and isogenicity of these NILs were confirmed with progeny tests and molecular marker analysis, respectively. Labelling studies demonstrated that the deficiency in GB accumulation was at the choline oxidation step.     

Characterization of maize lines differing in leaf abscisic Acid content in the field. 1 abscisic Acid physiology

The inbred maize lines Poljl7 and F-2 have previously been shown to differ by up to three-fold in leaf abscisic acid (ABA) concentration in the field. Lines from the cross Poljl7 × F-2 differing in leaf ABA concentrations, and the parents, were studied in the field to characterize the differences amongst the lines in ABA concentrations during the season, during the day and in different parts of the plants. The water status of the plants was measured and leaves were heat girdled to get information on possible causes for the genetic variation amongst the lines in ABA concentration. Leaf ABA concentrations of the high-AB A lines increased markedly and consistently from flowering time onwards, whereas leaf ABA concentrations of the low-ABA lines gradually fell after flowering. Leaf water potentials of high-ABA and low-ABA lines were similar during this time. Leaf ABA concentrations varied little during the day, and heat girdling caused a rise in ABA concentrations, which was similar in both high-ABA and low-ABA lines, only after girdling for at least 4 h. ABA concentrations were highest in the leaves and it was only in the leaves and developing kernels that substantial differences in ABA concentrations were found between the high-ABA and low-ABA classes. Although aerial brace roots also had high ABA concentrations, other roots and stem internodes had ABA concentrations which were consistently low and the same for both ABA classes. Differences between the ABA classes were unlikely to be due to differences in leaf water status or in ABA export from the leaves. Other possible explanations for the genotypic differences in leaf ABA concentrations are discussed.     

Photosynthesis is improved by exogenous glycinebetaine in salt-stressed maize plants

The effects of exogenous application of glycinebetaine (GB) (10 m M ) on growth, leaf water content, water use efficiency, photosynthetic gas exchange, and photosystem II photochemistry were investigated in maize plants subjected to salt stress (50 and 100 m M NaCl). Salt stress resulted in the decrease in growth and leaf relative water content as well as net photosynthesis and the apparent quantum yield of photosynthesis. Stomatal conductance, evaporation rate, and water use efficiency were decreased in salt-stressed plants. Salt stress also caused a decrease in the actual efficiency of PSII ( Φ _PSII), the efficiency of excitation energy capture by open PSII reaction centers ( F _v'/ F _m'), and the coefficients of photochemical quenching ( q _P) but caused an increase in non-photochemical quenching (NPQ). Salt stress showed no effects on the maximal efficiency of PSII photochemistry ( F _v/ F _m). On the other hand, in salt-stressed plants, GB application improved growth, leaf water content, net photosynthesis, and the apparent quantum yield of photosynthesis. GB application also increased stomatal conductance, leaf evaporation rate, and water use efficiency. In addition, GB application increased Φ _PSII, F _v'/ F _m', and q _P but decreased NPQ. However, GB application showed no effects on F _v/ F _m. These results suggest that photosynthesis was improved by GB application in salt-stressed plants and such an improvement was associated with an improvement in stomatal conductance and the actual PSII efficiency.     

Farnesol-like antitranspirant activity and stomatal behaviour in maize and Sorghum lines of differing drought tolerance

Abstract Potted seedlings of four lines of maize and Sorghum of differing drought tolerance were subjected to a single soil drying cycle and were only rewatered when the plants showed the first signs of wilting. Other plants remained well-watered throughout the experimental period. As plant water potentials decreased in the unwatered plants of three of the lines investigated (Sorghum Piper and M35-1, V-4146 and maize Farz 27), endogenous levels of farnesol-like antitranspirants increased. Closure of stomata correlated well with the increase in endogenous antitranspirant. In the fourth line (Sorghum M35-1, V-4184), stomata did not close as the level of plant water stress increased, although leaf diffusion resistance of even the well-watered plants of this line was quite high. In this line, there was no consistent relationship between plant water stress and antitranspirant level or between stomatal behaviour and antitranspirant level. The involvement of farnesol-like antitranspirants in the control of stomatal behaviour in water-stressed plants is discussed.     

Microbial expression profiles in the rhizosphere of two maize lines differing in N use efficiency

Plant and Soil - In Ontario, Canada, acreage of red clover (Trifolium pratense L.) intercropped with winter wheat (Triticum aestivum L. em. Thell) has declined, despite well-documented soil and...     

Analyses of genetic diversity among maize inbred lines differing for resistance to pink borer and post-flowering stalk rot

Identification of the diverse sources of resistance is an important issue among the breeders for developing pest and disease free hybrids, to reduce the inoculum load, to prolong the life of inbred lines/hybrids and to reduce the cost of cultivation. Molecular diversity analysis was carried out among 23 maize inbred lines with respect to post flowering stalk rot and pink borer. Forty six SSR markers were employed among eight post flowering stalk rot (PFSR) and seven pink borer resistant lines along with eight other inbred lines to identify diverse resistant sources for developing resistant heterotic combinations to above pests and diseases. Number of alleles per SSR marker ranged from 2 to 9 averaging 4.11. The polymorphism information content (PIC) ranged from 0.272 to 0.839 with an average of 0.568. Discrimination rate (DR) of the markers ranged from 0.095 to 0.861 with a mean of 0.618. Number of alleles was highly correlated with PIC and DR. The pair-wise genetic dissimilarity values ranged from 0.05 to 0.84 with an overall mean of 0.64. Un-weighted neighbour joining clustering put 23 genotypes in two main clusters, which were further subdivided into 5 and 6 sub-clusters, respectively. We obtained 56 rare and 26 unique alleles in specific inbred lines, which can be used for identification of these lines. The present study has revealed considerable diversity among inbred lines differing for resistance against PFSR and pink borer; and provided ample scope for selection of parents for utilization in heterosis breeding     

Hydraulic regulation strategies for whole-plant water balance of two maize inbred lines differing in drought resistance under short-term osmotic stress

The conservation of water in agriculture requires an understanding of the mechanisms of plant–water relations. This study aimed to reveal hydraulic regulation strategies of maize (Zea mays L.) for maintaining the plant water balance during drought. The water relations of two maize inbred lines (Tian4 and 478) that differ in their resistance to drought in the field were investigated under well-watered conditions and osmotic stress induced with 10 % PEG 6000. The leaf transpiration rate and leaf water potential of 478 varied diurnally, but remained constant in Tian4, which is more drought resistant. Tian4 plants showed morphological, anatomical and physiological advantages that protected them from foliar water loss. The strategies of leaf hydraulics to regulate leaf water balance during the day and during short-term osmotic stress also differed between Tian4 and 478. The leaf hydraulic conductivity of Tian4 and 478 increased temporarily, but their root hydraulic conductivities were reduced under osmotic stress. However, the root hydraulic conductivity of Tian4 subsequently recovered. Lower and rapidly reduced leaf transpiration and the ability of root hydraulics to recover from short-term osmotic stress can help explain the strategies for plant water balance of drought-tolerant maize.     

Inducers of glycinebetaine synthesis in barley.

Glycinebetaine is an osmoprotectant accumulated by barley (Hordeum vulgare) plants in response to high levels of NaCl, drought, and cold stress. Using barley seedlings in hydroponic culture, we characterized additional inducers of glycinebetaine accumulation. These included other inorganic salts (KCl, MgCl(2), LiCl, and Na(2)SO(4)), oxidants (H(2)O(2) and cumene hydroperoxide), and organic compounds (abscisic acid, polymixin B, n-butanol, salicylic acid, and aspirin). Stress symptoms brought on by high NaCl and other inducers, and not necessarily correlated with glycinebetaine accumulation, include wilting, loss of chlorophyll, and increase in thiobarbituric acid reacting substances. For NaCl, Ca(2+) ions at 10 to 20 mM decrease these stress symptoms without diminishing, or even increasing, glycinebetaine induction. Abscisic acid induces glycinebetaine accumulation without causing any of the stress symptoms. NaCl, KCl, and H(2)O(2) (but not other inducers) induce glycinebetaine at concentrations below those needed for the other stress symptoms. Mg(2+) at 10 to 20 mM induces both stress symptoms and glycinebetaine, but only at low (0.2 mM) Ca(2+). Although illumination is needed for optimal induction, a significant increase in the leaf glycinebetaine level is found in complete darkness, also.     

Co-expression of genes ApGSMT2 and ApDMT2 for glycinebetaine synthesis in maize enhances the drought tolerance of plants

Glycinebetaine plays an important role in the protection mechanism of many plants under various stress conditions. In this study, genetically engineered maize plants with an enhanced ability to synthesise glycinebetaine (GB) were produced by introducing two genes, glycine sarcosine methyltransferase gene (ApGSMT2) and dimethylglycine methyltransferase gene (ApDMT2), from the bacterium Aphanothece halophytica. Southern blotting and RT-PCR analysis demonstrated that the two genes were integrated into the maize genome and expressed. The increased expression levels of ApGSMT2 and ApDMT2 under drought conditions facilitated GB accumulation in the leaves of transgenic maize plants and conferred improved drought tolerance. Under drought conditions, the transgenic plants showed an increased accumulation of sugars and free amino acids, greater chlorophyll content, a higher photosynthesis rate and biomass, and lower malondialdehyde and electrolyte leakage compared to the wild-type; these results suggest that GB provides vital protection against drought stress. Under normal conditions, the transgenic plants did not show decreased biomass and productivity, which indicated that the co-expression of ApGSMT2 and ApDMT2 in maize plays an important role in its tolerance to drought stress and does not lead to detrimental effects. It was concluded that the co-expression of ApGSMT2 and ApDMT2 in maize is an effective approach to enhancing abiotic stress tolerance in maize breeding programmes.     

Adaptation to sub- and supraoptimal temperatures of inbred maize lines differing in origin with regard to seedling development and photosynthetic traits

Six inbred lines of maize ( Zea mays L.) from cool temperate regions (C) and from warm regions (W) were grown at 14, 22, 30 and 38°C up to the same physiological age, the full expansion of the third leaf. Generally, plants developed smaller shoot dry weights and leaf areas at extreme temperatures. The shoot:root ratio was lowest at 22°C and highest at 30°C. Most lines had a minimum for specific leaf dry weight at 30°C, but W lines had a second lower minimum at 14°C. Phosphofructokinase activity scarcely reacted to temperature between 22° and 38°C; at 14°C one C line and all W lines had rather low activities. Generally, the chlorophyll content increased steeply from 14 to 22°C and decreased somewhat from 30 to 38°C. In C lines the carotenoid level decreased from 14 to 38°C. No uniform temperature response was found for PEP carboxylase activity, but the highest activity was mostly attained at 38°C. RuBP carboxylase activity increased considerably from 14 to 22°C and remained comparatively constant at higher temperatures. The highest activity of NADP malate dehydrogenase was found at 22°C, with a decrease up to 38°C and with second lowest values at 14°C. C lines possessed larger leaf areas, shoot dry weights and higher shoot:root ratios than W lines at 14 and 22°C, and higher specific leaf dry weights over the whole temperature range. The genotypic pattern of shoot dry weight at 14°C corresponded reasonably well with that of phosphofructokinase activity. A better adaptation of C lines to suboptimal temperatures was mostly clearly indicated for photosynthetic traits which have a well proven relationship with the chloroplast membranes: chlorophyll, carotenoids and RuBP carboxylase. The least distinct effects of origin were observed at 38°C; a tendency prevailed for a better performance of C lines with regard to phosphofructokinase, carotenoids, RuBP carboxylase and NADP malate dehydrogenase.     

Complex duplications in maize lines

Rp1 is a disease resistance complex and is the terminal morphological marker on the short arm of maize chromosome 10. Several restriction fragment length polymorphisms (RFLPs), which map within 5 map units of Rp1, were examined to determine if they are also complex in structure. Two RFLP loci, which mapped distally to Rp1, BNL3.04 and PIO200075, existed in a single copy in all maize lines examined. These two loci cosegregated perfectly in 130 test cross progeny. Two RFLP loci that map proximally to Rp1 had unusual structures, which have not yet been reported for maize RFLPs; the loci were complex, with variable numbers of copies in different maize lines. One of the loci, NPI285, occasionally recombined in meiosis to yield changes in the number of copies of sequences homologous to the probe. The other proximal locus, detected by the probes NPI422, KSU3, and KSU4, was relatively stable in meiosis and no changes in the number of restriction fragments were observed. The similarity in map position between Rp1 and the complex RFLP loci indicate there may be genomic areas where variable numbers of repeated sequences are common. The structure of these complex loci may provide insight into the structure and evolution of Rp1.     

Meiotic behavior and DNA content in alloplasmic lines of maize.

The cytoplasm of Zea mays ssp. mexicana (teosinte) affects several inherited traits when combined with genotypes of maize (Zea mays ssp. mays). The meiotic behavior and the total DNA content of four lines of maize with teosinte cytoplasm were compared with those of the parental lines. The results obtained suggest that the cytoplasm of teosinte promotes an increase in total nuclear DNA content, perhaps through an increase of highly repetitive DNA in the knob zones. The analysis of meiotic behavior indicates that the cytoplasm of teosinte can alter the spatial distribution of the genomes, since two groups of five bivalents each were observed at a high frequency. During prophase I - anaphase I, each group of five bivalents behaves in a slightly asynchronous way with respect to the other group and, moreover, two nucleoli were observed in 10% of the cells. These results suggest that the cytoplasm of teosinte could induce changes affecting genomic structure and function in some maize genotypes. These changes are of potential importance for breeding programs and evolutionary studies.