Effects of temperature and light on the formation of chloroplast protrusions in leaf mesophyll cells of high alpine plants

Chloroplasts of many alpine plants have the ability to form marked, stroma-filled protrusions that do not contain thylakoids. Effects of temperature and light intensity on the frequency of chloroplasts with such protrusions in leaf mesophyll cells of nine different alpine plant species (Carex curvula All., Leontodon helveticus Merat., Oxyria digyna (L.) Hill., Poa alpina L. ssp. vivipara, Polygonum viviparum L., Ranunculus glacialis L., Ranunculus alpestris L., Silene acaulis L. and Soldanella pusilla Baumg.) covering seven different families were studied. Leaves were exposed to either darkness and a stepwise increase in temperature (10-38 degrees C) or to different light intensities (500 and 2000 micromol photons m(-2) s(-1)) and a constant temperature of 10 or 30 degrees C in a special temperature-regulated chamber. A chloroplast protrusions index characterising the relative proportion of chloroplasts with protrusions was defined. Seven of the nine species showed a significant increase in chloroplast protrusions when temperature was elevated to over 20 degrees C. In contrast, the light level did not generally affect the abundance of chloroplasts with protrusions. Chloroplast protrusions lead to a dynamic enlargement of the chloroplast surface area. They do not appear to be directly connected to a distinct photosystem II (PSII) (F(v)/F(m)) status and thus seem to be involved in secondary, not primary, photosynthetic processes.     

Changes in chloroplast ultrastructure in some high-alpine plants: adaptation to metabolic demands and climate?

Summary. The cytology of leaf cells from five different high-alpine plants was studied and compared with structures in chloroplasts from the typical high-alpine plant Ranunculus glacialis previously described as having frequent envelope plus stroma protrusions. The plants under investigation ranged from subalpine/alpine Geum montanum through alpine Geum reptans, Poa alpina var. vivipara, and Oxyria digyna to nival Cerastium uniflorum and R. glacialis. The general leaf structure (by light microscopy) and leaf mesophyll cell ultrastructure (by transmission electron microscopy [TEM]) did not show any specialized structures unique to these mountain species. However, chloroplast protrusion formation could be found in G. reptans and, to a greater extent, in O. digyna. The other species exhibited only a low percentage of such chloroplast structural changes. Occurrence of protrusions in samples of G. montanum and O. digyna growing in a mild climate at about 50 m above sea level was drastically reduced. Serial TEM sections of O. digyna cells showed that the protrusions can appear as rather broad and long appendices of plastids, often forming pocketlike structures where mitochondria and microbodies are in close vicinity to the plastid and to each other. It is suggested that some high-alpine plants may form such protrusions to facilitate fast exchange of molecules between cytoplasm and plastid as an adaptation to the short, often unfavorable vegetation period in the Alps, while other species may have developed different types of adaptation that are not expressed in ultrastructural changes of the plastids. Correspondence and reprints: Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Epidermal UV-screening of arctic and alpine plants along a latitudinal gradient in Europe

This study reports epidermal UV-transmittance in field-grown leaves of ecotypes of six species at three sites along a latitudinal UV-B gradient from Arctic Svalbard, via southern Norway to the French Alps for the years 1999–2001. Unexpectedly, Arctic populations had just as high epidermal UV-screening as alpine populations from lower latitudes. Dryas octopetala was the only species that significantly increased epidermal screening with increasing natural UV-B. Most species, however, showed clear differences in transmittance between years.Under controlled conditions in a growthroom, no ecotypic differences with respect to epidermal UV-B screening were found in Arctic and alpine ecotypes of Oxyria digyna, either in the absence or presence of UV-B radiation. Furthermore, UV-B transmittance in the absence of UV-B radiation in the growthroom was as low (5–6%) as in field-grown plants, indicating a high constitutive screening. Analysis of UV-B-absorbing phenolic compounds in O. digyna displayed no difference between the French Alps and Svalbard ecotypes, while the S. Norway ecotype contained significantly higher amounts of screening compounds. The qualitative analysis showed that the French Alps ecotype had a different composition of flavonoids compared with the two others, and that the ratio between di- and monohydroxylated flavonoids increased from south to north.     

4种北极被子植物叶片显微结构和超微结构研究

以采自北极的4种被子植物(高山发草Deschampsia alpina、无茎蝇子草Silene acaulis、仙女木Dryasocto-petala,极柳Salix polaris)为材料,利用光学显微镜和透射电子显微镜,研究了它们叶片显微结构和超微结构.结果显示;(1)高山发草和无茎蝇子草的叶片为等面叶,仙女木和极柳为两面叶,无茎蝇子草和极柳叶肉中含有异细胞;(2)4种植物的细胞超微结构均具有叶绿体紧贴细胞壁分布、叶绿体基质中含有淀粉粒、线粒体紧密地围绕在叶绿体周围的特征,而且高山发草和无茎蝇子草的类囊体出现膨胀,无茎蝇子草和仙女木的细胞基质有大量囊泡,极柳叶绿体内膜附近存在周质网.研究表明,北极植物叶片的结构特点与南极植物和高山植物具有一定的相似之处,北极植物叶片的显微结构和超微结构特征更有利于其适应北极的极端环境.     

Resource allocation patterns in a forb and a sedge in two arctic environments—short-term response to herbivory

The present work investigates C and N allocation patterns in two forage plants: a forb, Oxyria digyna , and a sedge, Eriophorum angustifolium , in subarctic Sweden and high arctic Canada. Short-term changes in concentrations after simulated or natural herbivory (caused by Gynaephora groenlandica on Oxyria in the high arctic habitat) were also investigated. There were no clear differences in concentrations of C and N between the high arctic and subarctic sites in either species. In Oxyria of the subarctic habitat, the minimum N concentrations occurred at earlier phenological stages compared with plants in the high arctic habitat. Simulated herbivory increased the concentration of C in belowground tissues relative to those in control plants in Oxyria at the subarctic site, which may indicate increased allocation of non-C compounds to the growing shoots or daughter ramets. Herbivory by Gynaephora groenlandica caterpillars increased the N concentrations of Oxyria both in aboveground and belowground tissues, possible indicating increased uptake of N in the high arctic habitat. Eriophorum did not show clear trends in concentrations relating to habitat, phenology or simulated herbivory. The difference between Oxyria and Eriophorum in their response to herbivory apparently resulted from contrasting growth habits between the species. Tiller death after reproduction and long leaf life span may be the main reasons for the lack of clear patterns in concentrations in Eriophorum. Compensation after herbivory may be attained by the early production of daughter tillers in Eriophorum instead of the regrowth of the damaged ramets, as in Oxyria. Monitoring the responses for only one season is apparently a too short time period in these long-lived plants.     

Refugia in the Cordilleran ice sheet of western North America: chloroplast DNA diversity in the Arctic–alpine plant Oxyria digyna

Aim  Late Pleistocene ice sheets are thought to have covered most of western Canada, including all of British Columbia (BC). We examine patterns of genetic variation in an Arctic–alpine plant to evaluate the possibility of full glacial refugia within the area covered by the Cordilleran ice sheet (CIS) and to uncover post-glacial migration routes.
Location  Western North America.
Methods  We sampled 1030 individuals of the Arctic–alpine plant Oxyria digyna from 117 populations distributed over much of its range in western and northern North America. DNA haplotypes were identified using restriction site analysis of two chloroplast DNA intergene spacer regions, psb A- trn H and trn T-L. We examined the geographical distribution of haplotype diversity in relation to latitude, and also compared various indices of diversity in putatively glaciated and unglaciated regions. Patterns of migration were inferred using nested clade analysis.
Results  We detected a total of 20 haplotypes. High haplotype diversity was found in Beringia, in unglaciated western USA, and in northern BC at 57–59° N, well within the accepted limits of the CIS. Ancestral haplotypes were also centred in northern BC.
Main conclusions  High genetic diversity of Oxyria digyna is expected in unglaciated regions, but unexpected in northern BC if British Columbia was entirely covered by ice during the late Pleistocene. Our observations suggest the presence of unglaciated areas providing late Pleistocene refugia in northern BC. Such refugia would have important implications for the origins and migrations of many plant and animal species in north-western North America.     

Shading-induced changes in the leaf mesophyll of plants of different functional types

Changes in the structural characteristics of mesophyll induced by shading were investigated in ten species of wild plants of diverse functional types. In all plant types, shading reduced leaf thickness and density by 30–50% and total surface of mesophyll, by 30–70%. The extent and mechanisms of mesophyll structural rearrangement depended on the plant functional type. In the ruderal plants, integral parameters of mesophyll, such as the surface of cells and chloroplasts and mesophyll resistance, changed threefold predominantly because of changes in the dimensions of the cells and chloroplasts. In these plants, shading reduced the volume of chloroplasts by 30%, and the chloroplast numbers per cell declined. The competitor plants showed a twofold increase in mesophyll resistance due to a decrease in the number of photosynthesizing cells per leaf area unit. Moreover, these plants maintained constant dimensions of mesophyll cells, ratios mesophyll surface/mesophyll volume and chloroplast surface/cell surface. In stress-tolerant plants, diffusion resistance of mesophyll remained the same irrespective of the growing conditions, and mesophyll rearrangement was associated with inversely proportional changes in the dimensions of the cells and cell volume per chloroplast. Noteworthy of these plants were relatively constant chloroplasts number per cell, per leaf area unit and total surface area of chloroplasts. The nature of relationship between the mesophyll diffusion resistance and structural parameters of leaf mesophyll differed in plants of diverse functional types.     

Central Asian origin of and strong genetic differentiation among populations of the rare and disjunct Carex atrofusca (Cyperaceae) in the Alps

Aim Carex atrofusca has an arctic–alpine distribution in the Northern Hemisphere, with only a few, disjunct localities known in the European Alps. These alpine populations are declining in number and size. In contrast, C. atrofusca has a wide circumpolar distribution range and is abundant in large parts of the Arctic. The degree of genetic differentiation of the alpine populations and their importance for the conservation of the intraspecific genetic variation of the species is unknown. Location Eurasia and Greenland, with emphasis on the European Alps. Methods We applied amplified fragment length polymorphism (AFLP) fingerprinting and sequences of chloroplast DNA to determine the position of the alpine populations in a circumpolar phylogeography of C. atrofusca and to unravel the patterns of genetic diversity and differentiation within the Alps. Results Two distinct major groups were detected in a neighbour‐joining analysis of AFLP data and in parsimony analysis of chloroplast DNA sequences: one consisting of the populations from Siberia and Greenland and one consisting of all European populations as sister to the populations from Central Asia. Within Europe, the populations from the Tatra Mountains and those from Scotland and Scandinavia formed two well‐supported groups, whereas the alpine populations did not constitute a group of their own. The genetic variation in the Alps was almost completely partitioned among the populations, and the populations were almost invariable. Main conclusions The alpine populations possibly originated due to immigration from Central Asia. The strong differentiation among them suggests that genetic drift has been strongly acting on the populations, either as a consequence of founder events during colonization or due to subsequent reduction of population sizes during warm stages of the Holocene.     

The impact of Pleistocene climate change on an ancient arctic-alpine plant: multiple lineages of disparate history in Oxyria digyna

The ranges of arctic-alpine species have shifted extensively with Pleistocene climate changes and glaciations. Using sequence data from the trnH-psbA and trnT-trnL chloroplast DNA spacer regions, we investigated the phylogeography of the widespread, ancient (>3 million years) arctic-alpine plant Oxyria digyna (Polygonaceae). We identified 45 haplotypes and six highly divergent major lineages; estimated ages of these lineages (time to most recent common ancestor, T(MRCA)) ranged from ～0.5 to 2.5 million years. One lineage is widespread in the arctic, a second is restricted to the southern Rocky Mountains of the western United States, and a third was found only in the Himalayan and Altai regions of Asia. Three other lineages are widespread in western North America, where they overlap extensively. The high genetic diversity and the presence of divergent major cpDNA lineages within Oxyria digyna reflect its age and suggest that it was widespread during much of its history. The distributions of individual lineages indicate repeated spread of Oxyria digyna through North America over multiple glacial cycles. During the Last Glacial Maximum it persisted in multiple refugia in western North America, including Beringia, south of the continental ice, and within the northern limits of the Cordilleran ice sheet. Our data contribute to a growing body of evidence that arctic-alpine species have migrated from different source regions over multiple glacial cycles and that cryptic refugia contributed to persistence through the Last Glacial Maximum.     

几种高山植物叶绿体淀粉粒的变化特征

利用透射电镜对生长于青藏高原东北部达坂山（海拔3900m）的5种高山植物叶绿体超微结构进行了观察。结果发现,在所研究的5种高山植物叶绿体中,淀粉粒数量均较多,淀粉粒呈长椭圆形或圆形,沿叶绿体长轴分布。在珠芽蓼的叶绿体中,淀粉粒的电子密度内外不均匀,外周电子密度低,中央电子密度高。在其余4种高山植物中,淀粉粒的电子密度均较低。另外,在这5种高山植物叶绿体中还出现了脂质小球。其类囊体均出现了不同程度的膨大现象。研究表明,高山植物叶绿体中淀粉粒的这种变化是对逆境的一种适应,是青藏高原特殊生态条件长期胁迫的结果。     

Divergence among arctic and alpine populations of the annual, Koenigia islandica: morphology, life-history, and phenology

Arctic and alpine habitats occur along complex environmental gradients, and over an extensive geographical range. Despite some selective forces common to these habitats, evolutionary divergence among populations of arctic and alpine plants along this gradient is expected. Of particular significance, both in the context of life-history theory and for implications of climate change, are the few annual species that have adapted to the constraints of an unpredictable, short growing season. In this study, morphological, life-history and phenological characters were found to differ significantly among six widely distributed populations of the arctic-alpine annual Koenigia islandica. On the basis of morphology and life-history traits, populations from high latitudes, with the exception of Svalbard, performed better in simulated arctic conditions, whereas the low latitude alpine plants from Colorado showed enhanced performance under simulated alpine conditions. On the basis of phenology, the six populations can be clearly grouped into arctic, high latitude alpine and alpine populations: arctic plants were found to develop and flower earliest; alpine plants latest. Because these results were obtained using seeds harvested from plants first grown through a complete generation in growth chambers, they indicate strong genetic differentiation. We discuss possible adaptive explanations for observed differences among the six geographically divergent populations.     

Photoinhibition, xanthophyll cycle and in vivo chlorophyll fluorescence quenching of chilling-tolerant Oxyria digyna and chilling-sensitive Zea mays

The relationship between susceptibility to photoinhibition, zeaxanthin formation and chlorophyll fluorescence quenching at suboptimal temperatures was studied in chilling-sensitive maize and in non-acclimated and cold-acclimated Oxyria digyna , a chilling-tolerant plant of arctic and alpine habitats. In maize, zeaxanthin formation was strongly suppressed by chilling. Zeaxanthin formed during preillumination at 20°C did not protect maize leaves from photoinhibition during a subsequent high-light, low-temperature treatment, as judged from the ratios of variable to maximal fluorescence, F_v/F_m. However, such preillumination significantly increased non-photochemical quenching (q_N) at low temperatures, mainly due to an enhancement of the fast-relaxing q_N component (i.e., of energy-dependent quenching. q_E). In O. digyna , cold-acclimation resulted in an increased zeaxanthin formation in the temperature range of 2.5–20°C. Cold-acclimation substantially decreased the susceptibility towards photoinhibition at 4°C, but q_N remained nearly unchanged between 2 and 38°C, as compared to control plants. Effects of cold acclimation on photosynthesis, photochemical quenching and quantum efficiency of photosystem II were small and indicated a slight amelioration only of the function of the photosynthetic apparatus at suboptimal temperatures (2–20°Ct. I) is concluded, that the xanthophyll cycle is strongly influenced by cold acclimation, while effects on the photosynthetic carbon assimilation only play a minor role in O. digyna.     

高山植物叶绿体与线粒体位置相关性研究

利用透射电镜对生长于青藏高原东北部达坂山(海拔3900m)的4种高山植物叶肉细胞进行了超微结构观察。首次在蒲公英(Taraxacum mongolicum)和乳白香青(Anaphalis lacten)的叶肉细胞中发现了叶绿体“吞噬”线粒体的现象。在所研究的4种高山植物中,线粒体的数量均较多,线粒体在细胞中的分布表现出不均一,且常分布在叶绿体附近,二者靠的很紧,常常可以观察到5～6个线粒体将叶绿体包围起来的现象。研究表明,高山植物叶肉细胞中叶绿体和线粒体在位置上的这种变化是对逆境的一种适应,是青藏高原特殊生态条件长期胁迫的结果。     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

High diversity of root associated fungi in both alpine and arctic <Emphasis Type="Italic">Dryas octopetala</Emphasis>

Background  

Dryas octopetala is a widespread dwarf shrub in alpine and arctic regions that forms ectomycorrhizal (ECM) symbiotic relationships with fungi. In this study we investigated the fungal communities associated with roots of D. octopetala in alpine sites in Norway and in the High Arctic on Svalbard, where we aimed to reveal whether the fungal diversity and species composition varied across the Alpine and Arctic regions. The internal transcribed spacer (ITS) region of nuclear ribosomal DNA was used to identify the fungal communities from bulk root samples obtained from 24 plants.     

Ultrastructural features of chilling-injury in Episcia reptans

Abstract. Chilling the leaves of the extremely chill-sensitive plant Episcia reptans for only a few hours at 5°C causes ultrastructural changes such as swelling and disorganization of chloroplasts and mitochondria, and vesiculation of cytoplasmic membranes. Rewarming plants after 5–6 h chilling causes further deterioration in chloroplast and mitochondrial structure and the appearance of fibrous material in the cytoplasm of epidermal, mesophyll and vascular cells. The nature of the deposit is not known but it may be the cause of the rapid development of chilling-injury on return of plants to the warmth.     

Bundle sheath chloroplasts of rice are more sensitive to drought stress than mesophyll chloroplasts

We investigated the effects of drought stress on the ultrastructure of chloroplasts in rice plants. After the seedlings were grown in a glasshouse for 1 month, they were treated for drought stress using two methods. One drought treatment was imposed by reducing the water supply to the plants for 1 month. The other was imposed by withholding water for 2 weeks to examine the withering process of leaves by drought stress. The ultrastructural changes of chloroplasts in bundle sheath cells were more prominent than those in mesophyll cells under both drought stress treatments. Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) content in bundle sheath chloroplasts reduced more dramatically than in mesophyll chloroplasts by drought stress. Although a slight swelling of thylakoids was sometimes observed in bundle sheath chloroplasts in moderate stress for 1 month, the thylakoids were less affected by drought stress than chloroplast envelope. These results suggest that chloroplasts in bundle sheath cells were more sensitive to drought stress than those in mesophyll cells and the thylakoids were less damaged by drought stress compared with chloroplast envelope.     

烟草NtFtsZ2-1基因参与叶绿体分裂和扩大的调节

叶绿体虽然是植物细胞内一种极其重要的细胞器,但其分裂的分子机制尚不很清楚。已经证明FtsZ蛋白作为真核细胞分裂装置的一个关键成分,参与叶绿体的分裂过程。烟草的FtsZ基因属于2个不同的家族,在对NtFtsZ1家族成员研究的基础上,用正义和反义表达技术研究了NtFtsZ2家族成员NtFtsZ2-1基因在转基因烟草中的功能。显微分析结果表明NtFtsZ2-1基因的表达水平异常增强或减弱都会严重干扰叶绿体的正常分裂过程,导致叶绿体在形态和数目上的异常（体积明显增大,数目显著减少）,而单个叶肉细胞中叶绿体的总表面积在正反义转基因烟草和野生型烟草之间保持了相对稳定,没有发生明显的变化。同时还证明NtFtsZ2-1基因表达的变化对叶绿素含量和叶绿体的光合作用能力没有直接的影响。据此我们认为NtFtsZ2-1基因参与叶绿体的分裂和体积的扩大,其表达水平的波动会改变植物中叶绿体的数目和大小,而且在叶绿体的数目与体积之间可能存在一种补偿机制,保证叶绿体能最大限度地吸收光能,从而使光合作用得以正常进行。     

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.