Thomas Björkman

Seed Treatment with Trichoderma harzianum Alleviates Biotic, Abiotic, and Physiological Stresses in Germinating Seeds and Seedlings

Trichoderma spp. are endophytic plant symbionts that are widely used as seed treatments to control diseases and to enhance plant growth and yield. Although some recent work has been published on their abilities to alleviate abiotic stresses, specific knowledge of mechanisms, abilities to control multiple plant stress factors, their effects on seed and seedlings is lacking. We examined the effects of seed treatment with T. harzianum strain T22 on germination of seed exposed to biotic stress (seed and seedling disease caused by Pythium ultimum) and abiotic stresses (osmotic, salinity, chilling, or heat stress). We also evaluated the ability of the beneficial fungus to overcome physiological stress (poor seed quality induced by seed aging). If seed were not under any of the stresses noted above, T22 generally had little effect upon seedling performance. However, under stress, treated seed germinated consistently faster and more uniformly than untreated seeds whether the stress was osmotic, salt, or suboptimal temperatures. The consistent response to varying stresses suggests a common mechanism through which the plant-fungus association enhances tolerance to a wide range of abiotic stresses as well as biotic stress. A common factor that negatively affects plants under these stress conditions is accumulation of toxic reactive oxygen species (ROS), and we tested the hypothesis that T22 reduced damages resulting from accumulation of ROS in stressed plants. Treatment of seeds reduced accumulation of lipid peroxides in seedlings under osmotic stress or in aged seeds. In addition, we showed that the effect of exogenous application of an antioxidant, glutathione, or application of T22, resulted in a similar positive effect on seed germination under osmotic stress or in aged seed. This evidence supports the model that T. harzianum strain T22 increases seedling vigor and ameliorates stress by inducing physiological protection in plants against oxidative damage.

Perception of Gravity by Plants

Physical principles can be used to predict some features about the gravity perception system in plants. The nature of the system has made it rather elusive, so this approach represents an additional source of information to help find it. For a gravitational stimulus to be detected, two masses must move relative to each other in a manner which causes a significant amount of work to be done on a receptor. Relative to cellular dimensions, the masses must be large, be dense and move noticeable distances. The main sources of noise are thermal motion and flexing of the plant tissue. Some new models for the function of amyloplasts as statoliths are presented.

The role of extracellular free-calcium gradients in gravitropic signalling in maize roots

Gravitropism in roots has been proposed to depend on a downward redistribution of calcium across the root cap. However, because of the many calcium-binding sites in the apoplast, redistribution might not result in a physiologically effective change in the apoplasmic calcium activity. To test whether there is such a change, we measured the effect of gravistimulation on the calcium activity of statocyte cell walls with calcium-specific microelectrodes. Such a measurement must be made on a tissue with gravity sensing cells at the surface. To obtain such a tissue, decapped maize roots (Zea mays L. cv. Golden Cross Bantam) were grown for 31 h to regenerate gravitropic sensitivity, but not root caps. The calcium activity in the apoplasm surrounding the gravity-sensing cells could then be measured. The initial pCa was 2.60 ± 0.28 (approx 2.5 mM). The calcium activity on the upper side of the root tip remained constant for 10 min after gravistimulation, then decreased 1.7-fold. On the lower side, after a similar lag the calcium activity increased 1.6-fold. Control roots, which were decapped but measured before recovering gravisensitivity (19 h), showed no change in calcium activity. To test whether this gradient is necessary for gravitropic curvature, we eliminated the calcium activity gradient during gravitropism by applying a mobile calcium-binding site (di-nitro-BAPTA; 1,2-bis(2-amino-5-nitro-phenoxy)ethane-N,N,N′,N′-tetraacetic acid) to the root cap; this treatment eliminated gravicurvature. A calcium gradient may be formed by proton-induced calcium desorption if there is a proton gradient. Preventing the formation of apoplastic pH gradients, using 10 and 50 mM 2-(N-morpholino)ethanesulfonic acid (Mes) buffer or 10 mM fusicoccin to stimulate proton excretion maximally, did not inhibit curvature; therefore the calcium gradient is not a secondary effect of a proton gradient. We have found a distinct and rapid differential in the apoplasmic calcium activity between the upper and lower sides of gravistimulated maize root tips which is necessary for gravitropism.

Trichoderma harzianum Enhances Antioxidant Defense of Tomato Seedlings and Resistance to Water Deficit

Some plant-symbiotic strains of the genus Trichoderma colonize roots and induce profound changes in plant gene expression that lead to enhanced growth, especially under biotic and abiotic stresses. In this study, we tested the hypothesis that one of the protective mechanisms enhanced by T. harzianum T22 colonization is the antioxidant defense mechanism. Having established that strain T22 modulates the expression of the genes encoding antioxidant enzymes, the status of antioxidant defense of tomato seedlings in response to colonization by T22 and water deficit was investigated. Total ascorbate or glutathione levels were not affected by either stimuli, but under water deficit, antioxidant pools became more oxidized (lower ratios of reduced to oxidized forms), whereas colonized plants maintained redox state as high as or higher than unstressed and untreated plants. The enhanced redox state of colonized plants could be explained by their higher activity of ascorbate and glutathione-recycling enzymes, higher activity of superoxide dismutase, catalase, and ascorbate peroxidase, in both root and shoot throughout the experiment. Similar enzymes were induced in uncolonized plants in response to water-deficit stress but to a lower extent when compared with colonized plants. This orchestrated enhancement in activity of reactive oxygen species (ROS)-scavenging pathways in colonized plants in response to stress supports the hypothesis that enhanced resistance of colonized plants to water deficit is at least partly due to higher capacity to scavenge ROS and recycle oxidized ascorbate and glutathione, a mechanism that is expected to enhance tolerance to abiotic and biotic stresses.

The effect of pollen load and pollen grain competition on fertilization success and progeny performance inFagopyrum esculentum

SummaryFlowers of cultivated buckwheat (Fagopyrum esculentum Moench) often receive natural pollen loads of fewer than 10 pollen grains. The cultivated varieties also have high genetic variability. These observations raise the question of whether seed production in buckwheat is often limited by pollen delivery, and whether small increases in pollen load could result in gametophytic selection through pollen grain competition. In greenhouse-grown buckwheat plants, embryo sac penetration by pollen tubes was universal with 10 or more pollen grains. However, seed production increased with pollen load up to 30 grains per flower. Larger pollen loads, which intensify selection among gametophytes, resulted in more vigorous progeny. Seedlings produced from high pollen load (15–20 pollen grains) were larger (40% by weight) than those from low pollen load (5 pollen grains). These results are evidence that pollen grain competition can occur in buckwheat with benefits for progeny performance.

The effectiveness of heterostyly in preventing illegitimate pollination in dish-shaped flowers

Heterostyly is thought to have evolved because it confers efficiency of cross-pollination. While it can be an alternative to an incompatibility mechanism in tube-shaped flowers, its effectiveness in dish-shaped flowers, such as those of Fagopyrum esculentum, has not been demonstrated. Stigmatic pollen loads were examined over the daily course of pollination in a planted field of this crop species. Natural pollination by bees resulted in 23% of the pollen on pin flowers and 69% on thrum flowers being legitimate. Both flower morphs therefore received the two pollen types in about the same proportions. Pin pollen constituted 74% of the pollen delivered to stigmata, even though the two morphs are present in equal numbers. Pin flowers collected about 75 pollen, and thrum flowers about 25 pollen by the end of the day of anthesis. The net result was about 15 compatible pollen per flower on both floral morphs. These results indicate that heterostyly is associated with excess pin-pollen delivery rather than excess legitimate-pollen delivery in F. esculentum.

Overcoming the barrier to interspecific hybridization of Fagopyrum esculentum with Fagopyrum tataricum

SummaryTartary buckwheat (Fagopyrum tataricum) was successfully hybridized with common buckwheat (F. esculentum), both diploid (2n=16), using the latter as male parent during bud pollination. The barrier normally encountered in such hybridization was overcome by enhancing the cross-compatibility of the two species, which was accomplished by synthesizing a unique genotype of common buckwheat. This novel plant was produced by selecting common buckwheat plants that exhibited, at isozyme loci PGM, SKDH and ADH, alleles with similar mobility to those found in tartary and then transferring these alleles to a single plant through six generations of breeding. Ovule culture was used to rescue the 7–10 day old embryos. On the rescue-culture medium 41% of the hybrid embryos formed calli larger than 200 μm in diameter. Most ceased to grow before reaching 1500 μm, but four out of 263 cultured ovules continued to grow as callus. One of these differentiated and formed callus with buds and shoots from which cloned plants were produced. The remaining embryos either did not grow at all or formed very small calli. When tartary was crossed with the original genotype of common buckwheat, only 22% of the hybrid embryos formed small calli and none differentiated. Hybridity of the calli and the plantlets was determined by RAPD and isozyme analysis, respectively. Flowers produced by the hybrid plants were of the same type (homomorphic) and size as those of tartary, but with white sepals like common buckwheat.

Variation in pollen performance among plants of Fagopyrum esculentum

SummaryIn genetically-heterogeneous outcrossing species, there is the opportunity for selection based on the male gamete. Buckwheat (Fagopyrum esculentum Moench) is self-incompatible with one ovule per flower, so pollen competition at each ovule can be studied. The occurrence of selection among pollen parents was determined, as well as the relative importance of prezygotic and post-zygotic selection. Mixed pollinations from two donors produced nonrandom paternity, with one of the donors being favored about 2:1 on several females. Individual plants showed significant variation in the speed of pollen-tube growth. Therefore, prezygotic selection is likely to have occurred based on the speed of pollen tube growth. In single-donor pollinations, donors had equal success as expected in the absence of post-zygotic selection among donors. However, a significant male x female interaction was found, consistent with postzygotic selection against particular parental combinations. To test whether male fitness is reduced by increased allocation to seed filling during pollen production, large- and small-seeded lines were compared, both as pollen donor and as pollen recipient. The large-seeded line was better in both roles, thus there was no evidence that greater allocation to seeds reduced the quality of the pollen.

Root growth regulation and gravitropism in maize roots does not require the epidermis

We have earlier published observations showing that endogenous alterations in growth rate during gravitropism in maize roots (Zea mays L.) are unaffected by the orientation of cuts which remove epidermal and cortical tissue in the growing zone (Björkman and Cleland, 1988, Planta 176, 513–518). We concluded that the epidermis and cortex are not essential for transporting a growth-regulating signal in gravitropism or straight growth, nor for regulating the rate of tissue expansion. This conclusion has been challenged by Yang et al. (1990, Planta 180, 530–536), who contend that a shallow girdle around the entire perimeter of the root blocks gravitropic curvature and that this inhibition is the result of a requirement for epidermal cells to transport the growth-regulating signal. In this paper we demonstrate that the entire epidermis can be removed without blocking gravitropic curvature and show that the position of narrow girdles does not affect the location of curvature. We therefore conclude that the epidermis is not required for transport of a growth-regulating substance from the root cap to the growing zone, nor does it regulate the growth rate of the elongating zone of roots.

Meristem identity gene expression during curd proliferation and flower initiation in Brassica oleracea

The regulation of reproductive development in cauliflower (Brassica oleracea var. botrytis DC) and broccoli (B. oleracea L. var. italica Plenck) is unusual in that most enlargement occurs while development is arrested at a distinct stage. Cauliflower and broccoli curds are composed of inflorescence meristems and flower buds, respectively. To determine whether this arrest is maintained by altered expression of the genes that specify these steps in Arabidopsis, the expression of each copy of their homologues (MADS-box genes BoAP1-a, BoAP1-c, BoCAL, BoFUL-a, BoFUL-b, BoFUL-c, and BoFUL-d; and non-MADS-box genes BoLFY, AP2, UFO, and BoTFL1) and the cauliflower curd-specific genes CCE1 and BoREM1 were measured simultaneously in heads that were arrested at different developmental stages by varying temperature, but had a common genotype. Transcript abundance of BoFUL paralogues and BoLFY was highest at the cauliflower stage of arrest, consistent with these genes initiating inflorescence meristems. The expression of other genes was the same regardless of the developmental stage of arrest. The expected models can therefore be excluded, wherein maintenance of arrest at the inflorescence meristem is a consequence of suppression of BoCAL, BoAP1-a, or BoLFY, or failure to suppress BoTFL1. Floral primordia and floral buds were normal in boap1-a boap1-c bocal triple mutants; therefore, other meristem identity genes can specify floral initiation (A-function) in B. oleracea. BoTFL1, a strong repressor of flowering in Arabidopsis, did not suppress the formation of the floral primordium in B. oleracea. Initiation of floral primordia and enlargement of floral buds in broccoli and cauliflower is not controlled solely by homologues of the genes that do so in Arabidopsis.