Comparative genetic study confirms exceptionally low genetic variation in the ancient and endangered relictual conifer,Wollemia nobilis (Araucariaceae)

The Wollemi pine, Wollemia nobilis (Araucariaceae), was discovered in 1994 as the only extant member of the genus, previously known only from the fossil record. With fewer than 100 trees known from an inaccessible canyon in southeastern Australia, it is one of the most endangered tree species in the world. We conducted a comparative population genetic survey at allozyme, amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) loci in W. nobilis, Araucaria cunninghamii and Agathis robusta - representatives of the two sister genera. No polymorphism was detected at 13 allozyme loci, more than 800 AFLP loci or the 20 SSR loci screened in W. nobilis. In Ag. robusta only one of 12 allozyme loci, five of 800 AFLP loci and none of the 15 SSR loci were variable. For A. cunninghamii, 10 of > 800 AFLP loci and five of 20 SSR loci were variable. Thus low genetic diversity characterizes all three species. While not ruling out the existence of genetic variation, we conclude that genetic diversity is exceptionally low in the Wollemi pine. To our knowledge this is the most extreme case known in plants. We conclude that the combination of small population effects, clonality and below-average genetic variation in the family are probable contributing factors to the low diversity. The exceptionally low genetic diversity of the Wollemi pine, combined with its known susceptibility to exotic fungal pathogens, reinforces current management policies of strict control of access to the pines and secrecy of the pine locations.     

Essential oil constituents derived from different organs of a relictual conifer Wollemia nobilis

The chemical composition of the essential oil of leaves (0.9%, w/v) and twigs (0.33%, w/v) of Wollemia nobilis (Araucariaceae) – a remnant species thought to have been extinct for 65 million years – was investigated by GC/MS. The main constituents of both leaf- and twig-derived oil samples were 16-kaurene (61.8% and 38.2%, respectively) and germacrene D (9.9% and 22%). The principal difference was a considerably more pronounced sesquiterpene presence in the twig-oil, amounting to 33.5%, than in its folial counterpart (23.4%). On the contrary, while remaining the dominant group in both oil samples under investigation, diterpenoids were relatively more abundant in leaf-derived oil constituting 65.3%, versus 41.7% detected in twigs. To our knowledge, this is the first report dealing with the essential oil composition of Wollemi pine twigs, as opposed to the leaf-derived volatiles.     

Axillary meristems and the development of epicormic buds in wollemi pine (Wollemia nobilis)

Intact trees of Wollemia nobilis Jones, Hill and Allen (Araucariaceae) routinely develop multiple coppice shoots as well as orthotropic epicormic shoots that become replacement or additional leaders. As these are unusual architectural features for the Araucariaceae, an investigation was made of the axillary meristems of the main stem and their role in the production of epicormic and possibly coppice shoots. Leaf axils, excised from the apex to the base of 2-m-high W. nobilis plants (seedling origin, ex situ grown), were examined anatomically. Small, endogenous, undifferentiated (no leaf primordia, no vascular or provascular connections) meristems were found in the axils from near the shoot apex. In the more proximal positions about half the meristems sampled did not differentiate further, but became tangentially elongated to compensate for increases in stem diameter. In the remaining axils the meristems slowly developed into bud primordia, although these buds usually developed few leaf primordia and their apical 'domes' were wide and flat. Associated vascular development was generally restricted to provascular dedifferentiation of the cortical parenchyma, with the procambium usually forming a 'closed loop' that did not extend back to the secondary vascular tissues. Development of the meristems was very uneven with adjacent axils often at widely differing stages of development into buds. The study shows that, unlike most conifers, W. nobilis possesses long-lived meristematic potential in most, if not all, leaf axils. Unlike other araucarias that have been investigated, many of the meristems in the orthotropic main stem will slowly develop into bud primordia beneath the bark in intact plants. It appears likely that this slow but continued development provides a ready source of additional or replacement leaders and thus new branches and leaves.     

Differences in proleptic and epicormic shoot structures in relation to water deficit and growth rate in almond trees (Prunus dulcis)

Background and Aims

Shoot characteristics differ depending on the meristem tissue that they originate from and environmental conditions during their development. This study focused on the effects of plant water status on axillary meristem fate and flowering patterns along proleptic and epicormic shoots, as well as on shoot growth rates on ‘Nonpareil’ almond trees (Prunus dulcis). The aims were (1) to characterize the structural differences between proleptic and epicormic shoots, (2) to determine whether water deficits modify shoot structures differently depending on shoot type, and (3) to determine whether shoot structures are related to shoot growth rates.

Methods

A hidden semi-Markov model of the axillary meristem fate and number of flower buds per node was built for two shoot types growing on trees exposed to three plant water status treatments. The models segmented observed shoots into successive homogeneous zones, which were compared between treatments. Shoot growth rates were calculated from shoot extension measurements made during the growing season.

Key Results

Proleptic shoots had seven successive homogeneous zones while epicormic shoots had five zones. Shoot structures were associated with changes in growth rate over the season. Water deficit (1) affected the occurrence and lengths of the first zones of proleptic shoots, but only the occurrence of the third zone was reduced in epicormic shoots; (2) had a minor effect on zone flowering patterns and did not modify shoot or zone composition of axillary meristem fates; and (3) reduced growth rates, although patterns over the season were similar among treatments.

Conclusions

Two meristem types, with different latency durations, produced shoots with different growth rates and distinct structures. Differences between shoot type structure responses to water deficit appeared to reflect their ontogenetic characteristics and/or resource availability for their development. Tree water deficit appeared to stimulate a more rapid progression through ontogenetic states.     

Micropropagation of licorice (Glycyrrhiza glabra L.) through shoot tip and nodal cultures

Summary Apical and axillary buds ofGlycyrrhiza glabra commonly known as licorice, a plant of repute in the Indian system of medicine, were used for induction of adventitious shoots. For induction of multiple shoots, Murashige and Skoog’s (MS) medium with N⁶-benzyladenine (BA, 0.88–8.87 μM) was used. Reduction in major salts of MS medium enhanced the multiplication ratio up to 1∶10. Plants transferred to the greenhouse showed 90% survival. The present work describes a stepwise protocol for production ofGlycyrrhiza glabra plants on simple minimal media, where very high multiplication rates with healthy root systems were obtained. Roots being the organ of commercial importance, the protocol has tremendous potential.     

Overexpression of a NAC-domain protein promotes shoot branching in rice

For a better understanding of shoot branching in rice (Oryza sativa), a rice activation-tagging library was screened for mutations in tiller development. Here, an activation-tagging mutant Ostil1 (Oryza sativa tillering1) was characterized, which showed increased tillers, enlarged tiller angle and semidwarf phenotype. Flanking sequence was obtained by plasmid rescue. RNA-interfering and overexpression transgenic rice plants were produced using Agrobacterium-mediated transformation. The mutant phenotype was cosegregated with the reallocation of Ds element, and the flanking region of the reallocated Ds element was identified as part of the OsNAC2 gene. Northern analysis showed that expression of OsNAC2 was greatly induced in the mutant plants. Transgenic rice overexpressing the OsNAC2 resulted in recapture of the mutant phenotype, while downregulation of OsNAC2 in the Ostil1 mutant through RNA interfering (RNAi) complemented the mutant phenotype, confirming that the Ostil1 was caused by overexpression of OsNAC2. Overexpression of OsNAC2 regulates shoot branching in rice. Overexpression of OsNAC2 contributes tiller bud outgrowth, but does not affect tiller bud initiation. This suggests that OsNAC2 has potential utility for improving plant structure for higher light-use efficiency and higher yield potential in rice.     

In vitro shoot multiplication of eastern white cedar (Thuja occidentalis)

Summary A protocol for clonal propagation of eastern white cedar (Thuja occidentalis L.) was enhanced by optimizing the shoot multiplication stage using unbranched in vitro-produced shoots. This was achieved by careful selection of different medium components. An optimum range of 10 to 14 axillary shoots was obtained when shoots were cultured on half-strength Quiorin and LePoivre medium containing 10μM filter-sterilized zeatin for 3 wk. Transfer of the treated shoots to cytokinin-free medium containing 0.05% activated charcoal improved both the number and quality of the axillary shoots produced. Maximum axillary bud induction was also accomplished when shoots were pulsed in 1 mM liquid, filter-sterilized zeatin for 3 h, and then transferred to half-strength Quiorin and LePoivre, charcoal-containing medium. Inclusion of 4% sucrose improved the number of axillary shoots obtained. Half strength of the major salts produced an optimum response. Shoots obtained from different cultures (1 to 5 yr old) responded similarly to the applied cytokinin; however, newly induced shoots (4 mo. old) gave a significantly higher response.     

Pattern and timing of diversification in Yucca (Agavaceae): specialized pollination does not escalate rates of diversification

The yucca-yucca moth interaction is one of the most well-known and remarkable obligate pollination mutualisms, and is an important study system for understanding coevolution. Previous research suggests that specialist pollinators can promote rapid diversification in plants, and theoretical work has predicted that obligate pollination mutualism promotes cospeciation between plants and their pollinators, resulting in contemporaneous, parallel diversification. However, a lack of information about the age of Yucca has impeded efforts to test these hypotheses. We used analyses of 4322 AFLP markers and cpDNA sequence data representing six non-protein-coding regions (trnT-trnL, trnL, trnL intron, trnL-trnF, rps16 and clpP intron 2) from all 34 species to recover a consensus organismal phylogeny, and used penalized likelihood to estimate divergence times and speciation rates in Yucca. The results indicate that the pollination mutualism did not accelerate diversification, as Yucca diversity (34 species) is not significantly greater than that of its non-moth-pollinated sister group, Agave sensu latissimus (240 species). The new phylogenetic estimates also corroborate the suggestion that the plant-moth pollination mutualism has at least two origins within the Agavaceae. Finally, age estimates show significant discord between the age of Yucca (ca 6-10Myr) and the current best estimates for the age of their pollinators (32-40Myr).     

A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro

Plant miRNAs, the critical regulator of gene expression, involve many development processes in vivo. However, the roles of miRNAs in plant cell proliferation and redifferntiation in vitro remain unknown. To determine better the molecular mechanism of these processes, we have recently reported that a set of miRNAs with different expression patterns between cells of totipotent and non-totipotent Arabidopsis calli. Some of these were specifically up- or downregulated during callus formation or shoot regeneration, and other development. Among them, miR160, and one of its target genes, ARF10, regulated Arabidopsis in vitro shoot regeneration via WUS, CLV3 and CUC1/2. The miR160-overexpressing, 35S transgenic lines, exhibited reduced shoot regeneration efficiency. The mARF10, a miR160-resistant form of ARF10, showed a high level of shoot regeneration ability. In the transgenic, expression of the above shoot meristem-specific genes was elevated, which is consistent with the improved shoot regeneration. In contrast, the ARF10 deficient knockout mutant produced fewer regenerated shoot. However, overexpressors of ARF10 were only marginally more efficient than the wild type with the respect to shoot regeneration. Our observation strongly supports that proper shoot regeneration from in vitro cultured cells requires the miR160-directed negative influence of ARF10. The enhanced expression of ARF10 is likely to have contributed to the improved regeneration ability.     

苹果MdPEPC基因家族鉴定及在腋芽萌发中的作用

磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxykinase,PEPC）家族蛋白普遍存在各种植物中,在光合碳同化过程中起到重要作用,同时具有多种非光合生物学功能,但PEPC基因在苹果（Malus domestica Borkh.）中尚未研究报道。本研究以苹果新基因组数据为基础,利用生物信息学方法对苹果PEPC家族成员（the members of apple PEPC family,MdPEPC）进行鉴定,并对其在不同组织中的表达谱以及去顶和细胞分裂素噻重氮苯基脲（thidazuron,TDZ）处理后苹果腋芽转录组中的表达模式进行分析,以期探究MdPEPC基因在参与苹果腋芽萌发中的作用。结果表明,苹果MdPEPC家族共有6个成员,分布于6条不同的染色体上,且理化特征较为相似;系统进化树及序列比对分析显示其可分为2个亚组（Group Ⅰ和Group Ⅱ）,其中Ⅰ组含4个MdPEPC家族成员,属于植物型PEPCs,而MdPEPC4和MdPEPC5则与拟南芥细菌型AtPPC4聚类到Ⅱ组;共线性分析表明,MdPEPC成员之间不存在串联重复,含7对片段重复;顺式作用元件分析显示,MdPEPC家族成员不仅受光和逆境等影响,还受多种激素综合调控;表达谱显示,除MdPEPC4和MdPEPC5外,其他植物型MdPEPC在不同组织中均有表达。转录组数据分析表明,去顶和TDZ处理后MdPEPC1和MdPEPC3表达量上调,而MdPEPC2则在处理后48 h明显下调表达。综上所述,本研究通过对苹果MdPEPC家族的鉴定和分析,筛选出MdPEPC1、MdPEPC2和MdPEPC3作为调控苹果腋芽萌发的候选基因,以便后期对其进行深入研究。     

First Record of Cosmocephalus obvelatus (Acuariidae) in Common Gulls (Larus canus) from Gangneung,Korea

A nematode species belonging to the genus Cosmocephalus was collected from the stomach of 2 common gulls, Larus canus. The common gulls were found dead on the seaside of Gangneung City, the Republic of Korea. The worms were identified and classified by light (LM) and scanning electron microscopy (SEM) on the basis of important taxonomic characters. The nematodes were characterized by a body length 9.1-9.3 mm (males) and 15.5-15.9 mm (females) and cordons recurrent in anterior direction and anastomosing laterally at about the level of anterior quarter of the buccal cavity. The salient bicuspid deirids were located on the posterior to the cordons. Lateral alae were well-developed, extending from the level just posterior of deirids to the level about middle of the body. LM and SEM observations identified the worms as C. obvelatus. This is the first reported case of C. obvelatus infection in common gulls in Korea.     

Annual and spatial variation in shoot demography associated with masting in Betula grossa: comparison between mature trees and saplings

Backgrounds and Aims

Shoot demography affects the growth of the tree crown and the number of leaves on a tree. Masting may cause inter-annual and spatial variation in shoot demography of mature trees, which may in turn affect the resource budget of the tree. The aim of this study was to evaluate the effect of masting on the temporal and spatial variations in shoot demography of mature Betula grossa.

Methods

The shoot demography was analysed in the upper and lower parts of the tree crown in mature trees and saplings over 7 years. Mature trees and saplings were compared to differentiate the effect of masting from the effect of exogenous environment on shoot demography. The fate of different shoot types (reproductive, vegetative, short, long), shoot length and leaf area were investigated by monitoring and by retrospective survey using morphological markers on branches. The effects of year and branch position on demographic parameters were evaluated.

Key Results

Shoot increase rate, production of long shoots, bud mortality, length of long shoots and leaf area of a branch fluctuated periodically from year to year in mature trees over 7 years, in which two masting events occurred. Branches within a crown showed synchronized annual variation, and the extent of fluctuation was larger in the upper branches than the lower branches. Vegetative shoots varied in their bud differentiation each year and contributed to the dynamic shoot demography as much as did reproductive shoots, suggesting physiological integration in shoot demography through hormonal regulation and resource allocation.

Conclusions

Masting caused periodic annual variation in shoot demography of the mature trees and the effect was spatially variable within a tree crown. Since masting is a common phenomenon among tree species, annual variation in shoot demography and leaf area should be incorporated into resource allocation models of mature masting trees.     

Hibernation does not affect memory retention in bats

Long-term memory can be critically important for animals in a variety of contexts, and yet the extreme reduction in body temperature in hibernating animals alters neurochemistry and may therefore impair brain function. Behavioural studies on memory impairment associated with hibernation have been almost exclusively conducted on ground squirrels (Rodentia) and provide conflicting results, including clear evidence for memory loss. Here, we for the first time tested memory retention after hibernation for a vertebrate outside rodents—bats (Chiroptera). In the light of the high mobility, ecology and long life of bats, we hypothesized that maintenance of consolidated memory through hibernation is under strong natural selection. We trained bats to find food in one out of three maze arms. After training, the pre-hibernation performance of all individuals was at 100 per cent correct decisions. After this pre-test, one group of bats was kept, with two interruptions, at 7°C for two months, while the other group was kept under conditions that prevented them from going into hibernation. The hibernated bats performed at the same high level as before hibernation and as the non-hibernated controls. Our data suggest that bats benefit from an as yet unknown neuroprotective mechanism to prevent memory loss in the cold brain.     

Heterodera achilleae n. sp. (Nematoda: Heteroderidae) from Yarrow in Yugoslavia

Heterodera achilleae n. sp., a member of the H. rostochiensis group, is described and illustrated from roots of yarrow, Achillea millefolium L. in Sarajevo, Yugoslavia. This new, round-cyst species differs from closely related species especially as follows: (1) from H. leptonepia, by having stouter larvae (a = 21), with longer styler (25 μ), and with outlet of dorsal esophageal gland averaging 5.7 μ from base o f styler; (2) from H. millefolii, in having excretory pore at base of neck and small, straight vulval slit of 5 μ; (3) from H. rostochiensis, in having a B/A ratio (Granek''s ratio) of 1.6 ; (4) from H. tabacum, by longer female stylet, two annules on female head, and males with outlet of dorsal esophageal gland further back (5.7 μ). In addition, H. achilleae n. sp. differs from the latter three species in having prominent longitudinal striae on the anterior half, or more, of cysts and females.     

Resistance to freezing in liquid nitrogen of carnation (Dianthus caryophyllus L. var Eolo) apical and axillary shoot tips excised from different aged in vitro plantlets

The ability of shoot tips from carnation (Dianthus caryophyllus L., var. Eolo) cultured in vitro to develop resistance to freezing in liquid nitrogen depends on the physiological state of the cell material and the pretreatment conditions. Regrowth rates close to 100% have been obtained with apical shoot tips isolated from 2 month-old stems, precultured on medium supplemented with sucrose (0.75M) and treated with dimethylsulfoxide (5% or more). Resistance of axillary shoot tips decreased progressively as a funtion of their distance from the apical shoot tip. During the development of the stem from axillary buds (obtained by cutting), progressive increases in the regrowth rate of frozen apices were noted, from 30% before cutting (axillary buds) to 98% after 3 weeks of culture.Abbreviations DMSO dimethylsulfoxide - LN liquid nitrogen     

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88 μM BAP with 108.6 μM adenine sulphate and 11.84 μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84 μM IBA, with 81.1 % rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.     

Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis

The cell cycle plays an important role in the development and adaptation of multicellular organisms; specifically, it allows them to optimally adjust their architecture in response to environmental changes. Kip-related proteins (KRPs) are important negative regulators of cyclin-dependent kinases (CDKs), which positively control the cell cycle during plant development. The Arabidopsis genome possesses seven KRP genes with low sequence similarity and distinct expression patterns; however, why Arabidopsis needs seven KRP genes and how these genes function in cell cycle regulation are unknown. Here, we focused on the characterization of KRP3, which was found to have unique functions in the shoot apical meristem (SAM) and leaves. KRP3 protein was localized to the SAM, including the ground meristem and vascular tissues in the ground part of the SAM and cotyledons. In addition, KRP3 protein was stabilized when treated with MG132, an inhibitor of the 26S proteasome, indicating that the protein may be regulated by 26S proteasome-mediated protein degradation. KRP3-overexpressing (KRP3 OE) transgenic plants showed reduced organ size, serrated leaves, and reduced fertility. Interestingly, the KRP3 OE transgenic plants showed a significant reduction in the size of the SAM with alterations in cell arrangement. In addition, compared to the wild type, the KRP3 OE transgenic plants had a higher DNA ploidy level in the SAM and leaves. Taken together, our data suggest that KRP3 plays important regulatory roles in the cell cycle and endoreduplication in the SAM and leaves.     

Post-pruning shoot growth increases fruit abscission and reduces stem carbohydrates and yield in macadamia

Background and Aims

There is good evidence for deciduous trees that competition for carbohydrates from shoot growth accentuates early fruit abscission and reduces yield but the effect for evergreen trees is not well defined. Here, whole-tree tip-pruning at anthesis is used to examine the effect of post-pruning shoot development on fruit abscission in the evergreen subtropical tree macadamia (Macadamia integrifolia, M. integrifolia × tetraphylla). Partial-tree tip-pruning is also used to test the localization of the effect.

Methods

In the first experiment (2005/2006), all branches on trees were tip-pruned at anthesis, some trees were allowed to re-shoot (R treatment) and shoots were removed from others (NR treatment). Fruit set and stem total non-structural carbohydrates (TNSC) over time, and yield were measured. In the second experiment (2006/2007), upper branches of trees were tip-pruned at anthesis, some trees were allowed to re-shoot (R) and shoots were removed from others (NR). Fruit set and yield were measured separately for upper (pruned) and lower (unpruned) branches.

Key Results

In the first experiment, R trees set far fewer fruit and had lower yield than NR trees. TNSC fell and rose in all treatments but the decline in R trees occurred earlier than in NR trees and coincided with early shoot growth and the increase in fruit abscission relative to the other treatments. In the second experiment, fruit abscission on upper branches of R trees increased relative to the other treatments but there was little difference in fruit abscission between treatments on lower branches.

Conclusions

This study is the first to demonstrate an increase in fruit abscission in an evergreen tree in response to pruning. The effect appeared to be related to competition for carbohydrates between post-pruning shoot growth and fruit development and was local, with shoot growth on pruned branches having no effect on fruit abscission on unpruned branches.     

Pseudaptinus (Thalpius) nobilis Liebke, new to the United States, and a key to the species of subgenus Thalpius LeConte in North America, including Mexico (Coleoptera, Carabidae, Zuphiini)

The Mexican carabid species Pseudaptinus (Thalpius) nobilis Liebke is documented from the United States for the first time based on two specimens captured in southeastern Texas. A new taxonomic key distinguishes the 10 members of subgenus Thalpius known in North America, including Mexico. Pseudaptinus (Thalpius) dorsalis, which is found to be highly variable in its dorsal coloration, is compared to similar Pseudaptinus (Thalpius) hoegei. Geographic ranges are extended for several species.     

Widespread introgression does not leak into allotopy in a broad sympatric zone

Species that overlap over a large part of their range and habitat requirements are challenging for the study of speciation and hybridization. In this respect, the study of broadscale introgressive hybridization has raised recent interest. Here we studied hybridization between two closely related amphibians Lissotriton helveticus and Lissotriton vulgaris that reproduce over a wide sympatric zone. We used mitochondrial and microsatellite markers on 1272 individuals in 37 sites over Europe to detect hybrids at the individual-level and to analyse Hardy-Weinberg and linkage disequilibria at the population-level. Morphological traits showed a strong bimodal distribution. Consistently, hybrid frequency was low (1.7%). We found asymmetric introgression with five times more hybrids in L. vulgaris than in L. helveticus, a pattern probably explained by an unequal effective population size in a study part wherein L. helveticus numerically predominates. Strikingly, significant levels of introgression were detected in 73% of sites shared by both species. Our study showed that introgression is widespread but remains confined to the sites where the two species reproduce at the same time. This pattern may explain why these species remain genetically distinct over a broad sympatric zone.