Pyruvate decarboxylase provides growing pollen tubes with a competitive advantage in petunia

Rapid pollen tube growth places unique demands on energy production and biosynthetic capacity. The aim of this work is to understand how primary metabolism meets the demands of such rapid growth. Aerobically grown pollen produce ethanol in large quantities. The ethanolic fermentation pathway consists of two committed enzymes: pyruvate decarboxylase ( PDC) and alcohol dehydrogenase ( ADH). Because adh mutations do not affect male gametophyte function, the obvious question is why pollen synthesize an abundant enzyme if they could do just as well without. Using transposon tagging in Petunia hybrida, we isolated a null mutant in pollen- specific Pdc2. Growth of the mutant pollen tubes through the style is reduced, and the mutant allele shows reduced transmission through the male, when in competition with wild- type pollen. We propose that not ADH but rather PDC is the critical enzyme in a novel, pollen- specific pathway. This pathway serves to bypass pyruvate dehydrogenase enzymes and thereby maintain biosynthetic capacity and energy production under the unique conditions prevailing during pollen - pistil interaction.

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1(S6)/ACT2(S6) double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1(S6) allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1(W138)/act1(S6) allele pair in a two-element dTph1 transposition system by producing 10 000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.

Microsurgical and laser ablation analysis of leaf positioning and dorsoventral patterning in tomato

Leaves are arranged according to regular patterns, a phenomenon referred to as phyllotaxis. Important determinants of phyllotaxis are the divergence angle between successive leaves, and the size of the leaves relative to the shoot axis. Young leaf primordia are thought to provide positional information to the meristem, thereby influencing the positioning of new primordia and hence the divergence angle. On the contrary, the meristem signals to the primordia to establish their dorsoventral polarity, which is a prerequisite for the formation of a leaf blade. These concepts originate from classical microsurgical studies carried out between the 1920s and the 1970s. Even though these techniques have been abandoned in favor of genetic analysis, the resulting insights remain a cornerstone of plant developmental biology. Here, we employ new microsurgical techniques to reassess and extend the classical studies on phyllotaxis and leaf polarity. Previous experiments have indicated that the isolation of an incipient primordium by a tangential incision caused a change of divergence angle between the two subsequent primordia, indicating that pre-existing primordia influence further phyllotaxis. Here.. we repeat these experiments and compare them with the results of laser ablation of incipient primordia. Furthermore. we explore to what extent the different pre-existing primordia influence the size and position of new organs. and hence phyllotaxis. We propose that the two youngest primordia (P-1 and P-2) are sufficient for the approximate positioning of the incipient primordium (I-1), and therefore for the perpetuation of the generative spiral, whereas the direct contact neighbours of I-1 (P-2 and P-3) control its delimitation and hence its exact size and position. Finally. we report L I specific cell ablation experiments suggesting that the meristem L-1 layer is essential for the dorsoventral patterning of leaf primordia.

Dissection of floral pollination syndromes in petunia

Animal-mediated pollination is essential in the reproductive biology of many flowering plants and tends to be associated with pollination syndromes, sets of floral traits that are adapted to particular groups of pollinators. The complexity and functional convergence of various traits within pollination syndromes are outstanding examples of biological adaptation, raising questions about their mechanisms and origins. In the genus Petunia, complex pollination syndromes are found for nocturnal hawkmoths (P. axillaris) and diurnal bees (P. integrifolia), with characteristic differences in petal color, corolla shape, reproductive organ morphology, nectar quantity, nectar quality, and fragrance. We dissected the Petunia syndromes into their most important phenotypic and genetic components. They appear to include several distinct differences, such as cell-growth and cell-division patterns in the basal third of the petals, elongation of the ventral stamens, nectar secretion and nectar sugar metabolism, and enzymatic differentiation in the phenylpropanoid pathway. In backcross-inbred lines of species-derived chromosome segments in a transposon tagging strain of P. hybrida, one to five quantitative trait loci were identified for each syndrome component. Two loci for stamen elongation and nectar volume were confirmed in introgression lines and showed large allelic differences. The combined data provide a framework for a detailed understanding of floral syndromes from their developmental and molecular basis to their impact on animal behavior. With its molecular genetic tools, this Petunia system provides a novel venue for a pattern of adaptive radiation that is among the most characteristic of flowering plants.

Domains of expansin gene expression define growth regions in the shoot apex of tomato

Expansins are members of a multigene family of extracellular proteins, which increase cell wall extensibility in vitro and thus are thought to be involved in cell expansion. The major significance of the presence of this large gene family may be that distinctly expressed genes can independently regulate cell expansion in place and time. Here we report on LeExp9, a new expansin gene from tomato, and compare its expression in the shoot tip with that of LeExp2 and LeExp18. LeExp18 gene is expressed in very young tissues of the tomato shoot apex and the transcript levels are upregulated in the incipient primordium. LeExp2 mRNA accumulated in more mature tissues and transcript levels correlated with cell elongation in the elongation zone. In situ hybridization experiments showed a uniform distribution of LeExp9 mRNA in submeristematic tissues. When gibberellin-deficient mutant tomatoes that lacked elongation of the internodes were treated with gibberellin, the phenotypic rescue was correlated with an increase in LeExp9 and LeExp2, but not LeExp18 levels. We propose that the three expansins define three distinct growing zones in the shoot tip. In the meristem proper, gibberellin-independent LeExp18 mediates the cell expansion that accompanies cell division. In the submeristematic zone, LeExp9 mediates cell expansion at a time that cell division comes to a halt. LeExp9 expression requires gibberellin but the hormone is not normally limiting. Finally, LeExp2 mediates cell elongation in young stem tissue. LeExp2 expression is limited by the available gibberellin. These data suggest that regulation of cell wall extensibility is controlled, at least in part, by differential regulation of expansin genes.

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al ., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of C-14-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.

Evidence of species-specific neighborhood effects in the dipterocarpaceae of a Bornean rain forest

Although accumulating evidence indicates that local intraspecific density-dependent effects are not as rare in species-rich communities as previously suspected, there are still very few detailed and systematic neighborhood analyses of species-rich communities. Here, we provide such an analysis with the overall goal of quantifying the relative importance of inter- and intraspecific interaction strength in a primary, lowland dipterocarp forest located at Danum, Sabah, Malaysia. Using data on 10 abundant overstory dipterocarp species from two 4-ha permanent plots, we evaluated the effects of neighbors on the absolute growth rate of focal trees (from 1986 to 1996) over increasing neighborhood radii (from 1 to 20 m) with multiple regressions. Only trees 10 cm to < 100 cm girth at breast height in 1986 were considered as focal trees. Among neighborhood models with one neighbor term, models including only conspecific larger trees performed best in five out of 10 species. Negative effects of conspecific larger neighbors were most apparent in large overstory species such as those of the genus Shorea. However, neighborhood models with separate terms and radii for heterospecific and conspecific neighbors accounted for more variability in absolute growth rates than did neighborhood models with one neighbor term. The conspecific term was significant for nine out of 10 species. Moreover, in five out of 10 species, trees without conspecific neighbors had significantly higher absolute growth rates than trees with conspecific neighbors. Averaged over the 10 species, trees without conspecific neighbors grew 32.4 cm(2) in basal area from 1986 to 1996, whereas trees with conspecific neighbors only grew 14.7 cm(2) in basal area, although there was no difference in initial basal area between trees in the two groups. Averaged across the six species of the genus Shorea, negative effects of conspecific larger trees were significantly stronger than for heterospecific larger neighbors. Thus, high local densities within neighborhoods of 20 m may lead to strong intraspecific negative and, hence, density-dependent, effects even in species rich communities with low overall densities at larger spatial scales. We conjecture that the strength of conspecific effects may be correlated with the degree of host specificity of ectomycorrhizae.

Secondary succession and dipterocarp recruitment in Bornean rain forest after logging

To understand succession in dipterocarp rain forest after logging, the structure, species composition and dynamics of primary (PF) and secondary (SF) forest at Danum were compared. In 10 replicate 0.16-ha plots per forest type trees >= 10 cm gbh (3.2 cm dbh) were measured in 1995 and 2001. The SF had been logged in 1988, which allowed successional change to be recorded at 8 and 13 years. In 2001, saplings (1.0-3.1 cm dbh) were measured in nested quadrats. The forest types were similar in mean radiation at 2 m height, and in density, basal area and species number of all trees. Among small (10 <= 31.4) and large ( >= 31.4 cm gbh) trees, in both 1995 and 2001, there were 10- and 3-fold more dipterocarps in SF than PF respectively; and averaging over the two dates, there were correspondingly ca. 10- and 18-fold more pioneers. Mortality was ca. 60% higher in SF than PF, largely due to a seven-fold difference for pioneers: for dipterocarps there was little difference. Recruitment was similar in PF and SE Stem growth rates were 37% higher in SF than PF for all trees, although dipterocarps showed the opposite trend. Among saplings, dipterocarps dominated SF with a 10-fold higher density than in PF. For dipterocarps, the light (LH) and medium-heavy (MHH) canopy hardwoods, and the shade-tolerant, smaller-stature other (OTH) species (e.g. Hopea and Vatica) were in the ratios ca. 40:15:45 in SF and 85: < 1:15 in PF. LHs had higher mortality than OTHs in SE In PF ca. 80% of the saplings were LH: in SF ca. 70% were OTH. The predominance of OTHs in SF is explained by the logging of primary rain forest which was in a likely late stage of recovery from natural disturbance, plus the continuing shaded conditions in the understorey promoted by dense pioneer vegetation. At 13 years after logging succession appeared to be inhibited: LHs were being suppressed but MHHs and OTHs persisted. Succession in lowland dipterocarp, rain forests may therefore depend on the successional state of the primary forest when it is logged. A review of logged versus unlogged studies in Borneo highlights the need for more detailed ecological comparisons.

Resistance of a lowland rain forest to increasing drought intensity in Sabah, Borneo

Occasional strong droughts are an important feature of the climatic environment of tropical rain forest in much of Borneo. This paper compares the response of a lowland dipterocarp forest at Danum, Sabah, in a period of low (LDI) and a period of high (HDI) drought intensity (1986-96, 9.98 y;1996-99, 2.62 y). Mean annual drought intensity was two-fold higher in the HDI than LDI period (1997 v. 976 mm), and each period had one moderately strong main drought (viz. 1992, 1998). Mortality of `all' trees greater than or equal to 10 cm gbh (girth at breast height) and stem growth rates of `small' trees 10less than or equal to50 cm gbh were measured in sixteen 0.16-ha subplots (half on ridge, half on lower slope sites) within two 4-ha plots. These 10-50-cm trees were composed largely of true understorey species. A new procedure was developed to correct for the effect of differences in length of census interval when comparing tree mortality rates. Mortality rates of small trees declined slightly but not significantly between the LDI and HDI periods (1.53 to 1.48% y(-1)): mortality of all trees showed a similar pattern. Relative growth rates declined significantly by 23% from LDI to HDI periods (11.1 to 8.6 mm m(-1) y(-1)): for absolute growth rates the decrease was 28% (2.45 to 1.77 mm y(-1)). Neither mortality nor growth rates were significantly influenced by topography. For small trees, across subplots, absolute growth rate was positively correlated in the LDI period, but negatively correlated in the HDI period, with mortality rate. There was no consistent pattern in the responses among the 19 most abundant species (n greater than or equal to 50 trees) which included a proposed drought-tolerant guild. In terms of tree survival, the forest at Danum was resistant to increasing drought intensity, but showed decreased stem growth attributable to increasing water stress.

The dating of dipterocarp tree rings: establishing a record of carbon cycling and climatic change in the tropics

In a first step to obtain a proxy record of past climatic events (including the El Ni (n) over tildeo-Southern Oscillation) in the normally aseasonal tropical environment of Sabah, a radial segment from a recently fallen dipterocarp (Shorea Superba) was radiocarbon dated and subjected to carbon isotope analysis. The high-precision radiocarbon results fell into the ambiguous modern plateau where several calibrated dates can exist for each sample. Dating was achieved by wiggle matching using a Bayesian approach to calibration. Using the defined growth characteristics of Shorea superba, probability density distributions were calculated and improbable dates rejected. It was found that the tree most likely started growing around AD 1660-1685. A total of 173 apparent growth increments were measured and, therefore, it could be determined that the tree formed one ring approximately every two years. Stable carbon isotope values were obtained from resin-extracted wholewood from each ring. Carbon cycling is evident in the `juvenile effect', resulting from the assimilation of respired carbon dioxide and lower light levels below the canopy, and in the `anthropogenic effect' caused by increased industrial activity in the late-nineteenth and twentieth centuries. This study demonstrates that palaeoenvironmental information can be obtained from trees growing in aseasonal environments, where climatic conditions prevent the formation of well-defined annual rings.

Structure and inferred dynamics of a large grove of Microberlinia bisulcata trees in central African rain forest: the possible role of periods of multiple disturbance events

A 272-ha grove of dominant Microberlinia bisulcata (Caesalpinioideae) adult trees greater than or equal to 50 cm stem diameter was mapped in its entirety in the southern part of Korup National Park, Cameroon. The approach used an earlier-established 82.5-ha permanent plot with a new surrounding 50-m grid of transect lines. Tree diameters were available from the plot but trees on the grid were recorded as being greater than or equal to 50 cm. The grove consisted of 1028 trees in 2000. Other species occurred within the grove. including the associated subdominants Tetraberlinia bifoliolata and T. korupensis. Microberlinia bisulcata becomes adult at a stein diameter of c. 50 cm and at an estimated age of 50 y. Three oval-shaped subgroves with dimensions c. 8 50 in x 13 50 in (90 ha) were defined. For two of them (within the plot) tree diameters were available. Subgroves differed in their scales and intensities of spatial tree patterns, and in their size frequency distributions, these suggesting differing past dynamics. The modal scale of clumping was 40-50 m. Seed dispersal by pod ejection (to c. 50 in) was evident from the semi-circles of trees at the grove's edge and from the many internal circles (100-200 m diameter). The grove has the capacity. therefore, to increase at c. 100 m per century. To form its present extent and structure. it is inferred that it expanded and infilled from a possibly smaller area of lower adult-tree density. This possibly happened in three waves of recruitment, each one determined by a period of several intense disturbances. Climate records for Africa show that 1740-50 and 1820-30 were periods of drought, and that 1870-1895 was also regionally very dry. Canopy openings allow the light-demanding and fast-growing ectomycorrhizal M. bisulcata to establish, but successive releases are thought to be required to achieve effective recruitment. Nevertheless, in the last 50 y there were no major events and recruitment in the grove was very poor. This present study leads to a new hypothesis of the role of periods of multiple extreme events being the driving factor for the population dynamics of many large African tree species such as M. bisulcata.

Rainfall input, throughfall and stemflow of nutrients in a central African rain forest dominated by ectomycorrhizal trees

Incident rainfall is a major source of nutrient input to a forest ecosystem and the consequent throughfall and stemflow contribute to nutrient cycling. These rain-based fluxes were measured over 12 mo in two forest types in Korup National Park, Cameroon, one with low (LEM) and one with high (HEM) ectomycorrhizal abundances of trees. Throughfall was 96.6 and 92.4% of the incident annual rainfall (5370 mm) in LEM and HEM forests respectively; stemflow was correspondingly 1.5 and 2.2%. Architectural analysis showed that ln(funneling ratio) declined linearly with increasing ln(basal area) of trees. Mean annual inputs of N, P, K, Mg and Ca in incident rainfall were 1.50, 1.07, 7.77, 5.25 and 9.27 kg ha(-1), and total rain-based inputs to the forest floor were 5.0, 3.2, 123.4, 14.4 and 37.7 kg ha-1 respectively. The value for K is high for tropical forests and that for N is low. Nitrogen showed a significantly lower loading of throughfall and stemflow in HEM than in LEM forest, this being associated in the HEM forest with a greater abundance of epiphytic bryophytes which may absorb more N. Incident rainfall provided c. 35% of the gross input of P to the forest floor (i. e., rain-based plus small litter inputs), a surprisingly high contribution given the sandy P-poor soils. At the start of the wet season leaching of K from the canopy was particularly high. Calcium in the rain was also highest at this time, most likely due to washing off of dry-deposited Harmattan dusts. It is proposed that throughfall has an important `priming' function in the rapid decomposition of litter and mineralization of P at the start of the wet season. The contribution of P inputted from the atmosphere appears to be significant when compared to the rates of P mineralization from leaf litter.

The role of tree size in the leafing phenology of a seasonally dry tropical forest in Belize, Central America

Leafing phenology of two dry-forest sites on soils of different depth (S = shallow, D = deep) at Shipstern Reserve, Belize, were compared at the start of the rainy season (April-June 2000). Trees greater than or equal to 2.5 cm dbh were recorded weekly for 8 wk in three 0.04-ha plots per site. Ten species were analysed individually for their phenological patterns, of which the three most common were Bursera simaruba, Metopium brownei and Jatropha gaumeri. Trees were divided into those in the canopy (> 10 cm dbh) and the subcanopy (less than or equal to 10 cm dbh). Site S had larger trees on average than site D. The proportion of trees flushing leaves at any one time was generally higher in site S than in site D, for both canopy and subcanopy trees. Leaf flush started 2 wk earlier in site S than site D for subcanopy trees, but only 0.5 wk earlier for the canopy trees. Leaf flush duration was 1.5 wk longer in site S than site D. Large trees in the subcanopy flushed leaves earlier than small ones at both sites but in the canopy just at site D. Large trees flushed leaves earlier than small ones in three species and small trees flushed leaves more rapidly in two species. Bursera and Jatropha followed the general trends but Metopium, with larger trees in site D than site S, showed the converse with onset of flushing I wk earlier in site D than site S. Differences in response of the canopy and subcanopy trees on each site can be accounted for by the predominance of spring-flushing or stem-succulent species in site S and a tendency for evergreen species to occur in site D. Early flushing of relatively larger trees in site D most likely requires access to deeper soil water reserves but small and large trees utilize stored tree water in site S.

Drought avoidance and the effect of local topography on trees in the understorey of Bornean lowland rain forest

The water relations of two tree species in the Euphorbiaceae were compared to test in part a hypothesis that the forest understorey plays an integral role in drought response. At Danum, Sabah, the relatively common species Dimorphocalyx muricatus is associated with ridges whilst another species, Mallotus wrayi, occurs widely both on ridges and lower slopes. Sets of subplots within two 4 -ha permanent plots in this lowland dipterocarp rain forest, were positioned on ridges and lower slopes. Soil water potentials were recorded in 1995-1997, and leaf water potentials were measured on six occasions. Soil water potentials on the ridges (-0.047 MPa) were significantly lower than on the lower slopes (-0.012 MPa), but during the driest period in May 1997 they fell to similarly low levels on both sites (-0.53 MPa). A weighted 40-day accumulated rainfall index was developed to model the soil water potentials. At dry times, D. muricatus (ridge) had significantly higher pre-dawn (-0.21 v. -0.57 MPa) and mid-day (-0.59 v. -1.77 MPa) leaf water potentials than M. wrayi (mean of ridge and lower slope). Leaf osmotic potentials of M. wrayi on the ridges were lower (-1.63 MPa) than on lower slopes (-1.09 MPa), with those for D. muricatus being intermediate (-1.29 MPa): both species adjusted osmotically between wet and dry times. D. muricatus trees were more deeply rooted than M. wrayi trees (97 v. 70 cm). M. wrayi trees had greater lateral root cross-sectional areas than D. muricatus trees although a greater proportion of this sectional area for D. muricatus was further down the soil profile. D. muricatus appeared to maintain relatively high water potentials during dry periods because of its access to deeper water supplies and thus it largely avoided drought effects, but M. wrayi seemed to be more affected yet tolerant of drought and was more plastic in its response. The interaction between water availability and topography determines these species' distributions and provides insights into how rain forests can withstand occasional strong droughts.