番荔枝科植物叶的比较解剖学

利用扫描电镜、叶表皮离析法和石蜡切片法研究了番荔枝科93种2变种植物叶片的形态结构.结果表明番荔枝科植物叶片形态结构具有较大相似性,如叶表面均具有表皮毛,表皮细胞具有晶体,气孔器为平列型,具2～6个副卫细胞,仅分布在远轴面,普遍具有败育气孔器,叶肉组织中普遍含有油细胞等,但表皮毛的类型,表皮细胞的形状,表皮细胞内晶体的类型和形态,叶肉组织的结构具有明显的属间和种间差异.     

Nanolitre-scale assays to determine the activities of enzymes in individual plant cells

There are a variety of methods for characterising gene expression at the level of individual cells and for demonstrating that the cells also contain the encoded proteins. However, measuring the activity of enzymes at the resolution of single cells in complex tissues, such as leaves, is problematic. We have addressed this by using single-cell sampling to extract 10-100 pl droplets of sap from individual plant cells and then measuring enzyme activities in these droplets with nanolitre-scale fluorescence-based assays. We have optimised these assays and used them to measure and characterise the activities of acid phosphatase, cysteine protease and nitrate reductase in sap samples from epidermal and mesophyll cells of barley (Hordeum vulgare L.) and Arabidopsis thaliana leaves exposed to different developmental and environmental conditions. During leaf senescence in barley, we found that the dynamics with which acid phosphatase and protease activities changed were different in each cell type and did not mirror the changes occurring at the whole-leaf level. Increases in nitrate reductase activities after exposure of barley plants to nitrate were large in mesophyll cells but small in epidermal cells. The technique was applied successfully to Arabidopsis and, as in barley, revealed cell-specific differences in the activities of both acid phosphatase and nitrate reductase. The assays add to the spectrum of techniques available for characterising cells within complex plant tissues, thus extending the opportunity to relate gene expression to biochemical activities at the single-cell level.     

柠条锦鸡儿小叶解剖结构的环境分异性研究

以巴丹吉林、腾格里、乌兰布和及库布齐沙漠的柠条锦鸡儿成熟叶片为研究对象,采用石蜡切片法,观察叶的横切结构,使用Motic Images Plus 2测定叶片厚度、栅栏组织细胞长和上、下表皮厚度等指标,分析比较4个沙漠的柠条锦鸡儿小叶解剖结构差异。结果表明:(1)生长环境不同,柠条锦鸡儿小叶在表皮、叶肉和叶脉结构上都存在差异,以巴丹吉林沙漠与库布齐沙漠的差异性最为显著。(2)干旱环境下,叶片变小、变厚,栅栏组织和维管束组织更为发达,巴丹吉林沙漠最为干旱,叶片最厚,栅栏组织细胞层数最多、细胞最长,导管列数最多;库布齐沙漠水分条件最好,叶片最薄,栅栏组织细胞最短且层数最少,导管列数最少。(3)对柠条锦鸡儿小叶解剖结构的各项指标与环境参数进行相关分析发现,温度和降水是影响柠条锦鸡儿小叶解剖结构变化的主要因素。     

番荔枝科蚁花属和澄广花属叶的比较解剖学研究

利用扫描电镜技术,叶片离析方法和石蜡切片法对蚁花属1种和澄广花属9种植物叶的形态结构进行比较研究。结果表明,两属植物有许多相似之处,但又有以下一些显著不同;蚁花属植物叶表皮细胞均具一晶族,叶肉组织中具1-2层栅栏组织细胞,油细胞均匀分布在栅栏组织和海绵组织中,栅栏组织在主脉处不连续,而澄广花属植物叶的表皮细胞内具一单斜晶,叶肉组织中具1层栅栏组织细胞,油细胞仅分布在海绵组织中,栅栏组织在主脉处连续,结果为蚁花属和澄广花属的分类学处理提供了新证据。     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

A soybean (<Emphasis Type="Italic">Glycine max</Emphasis>) polyubiquitin promoter gives strong constitutive expression in transgenic soybean

The success of plant genetic transformation relies greatly on the strength and specificity of the promoters used to drive genes of interest. In this study, we analyzed gfp gene expression mediated by a polyubiquitin promoter (Gmubi) from soybean (Glycine max) in stably transformed soybean tissues. Strong GFP expression was observed in stably transformed proliferative embryogenic tissues. In whole transgenic plants, GFP expression was observed in root tips, main and lateral roots, cotyledons and plumules in young plants as well as in leaf veins, petioles, flower petals, pollen, pods and developing seeds in mature plants. GFP expression was localized mainly in epidermal cells, leaf mesophyll, procambium and vascular tissues. Introduction of an intron-less version of the Gmubi promoter (Gmupri) displayed almost the same GFP expression pattern albeit at lower intensities. The Gmubi promoter showed high levels of constitutive expression and represents an alternative to viral promoters for driving gene expression in soybean.     

山东银莲花叶片形态结构对异质生境和海拔变化的响应

山东银莲花为一分布极其狭域的稀有物种,对海拔600 m以上的针阔混交林和山顶灌丛两种异质的生境都具有较高的适应性。为探索其适应策略,选择两种异质生境中的5个海拔梯度样带,采用常规石蜡切片法和显微观察技术,对叶片进行观察、分析和测量,通过比较叶片外部形态特征参数和内部解剖结构的差异,推测其叶片适应海拔和异质生境的响应策略。结果表明:为适应阴暗、潮湿的针阔混交林和干旱、强光照的山顶灌丛两种不同环境,山东银莲花分别表现出不同的适应策略。针阔混交林下,叶片的背腹表皮毛密度、比叶面积和气孔相对开度较山顶灌丛的大,而气孔密度、叶片厚度、栅栏组织和海绵组织的厚度较山顶灌丛的小;山顶灌丛植株叶片栅栏组织细胞排列较林下更加整齐紧密。两种生境中叶片腹面表皮毛的长度、气孔相对开度都随海拔的升高而减小,且差异明显;而叶片厚度、比叶面积、气孔指数等对600 m以上海拔变化未表现出明显的规律性。本研究将为山东银莲花的保护和利用提供理论基础及依据,为其他植物的相关研究提供参考。     

Subcellular localisation of Cd and Zn in the leaves of a Cd-hyperaccumulating ecotype of<Emphasis Type="Italic"> Thlaspi caerulescens</Emphasis>

Thlaspi caerulescens (Ganges ecotype) is able to accumulate large concentrations of cadmium (Cd) and zinc (Zn) in the leaves without showing any toxicity, suggesting a strong internal detoxification. The distribution of Cd and Zn in the leaves was investigated in the present study. Although the Cd and Zn concentrations in the epidermal tissues were 2-fold higher than those of mesophyll tissues, 65–70% of total leaf Cd and Zn were distributed in the mesophyll tissues, suggesting that mesophyll is a major storage site of the two metals in the leaves. To examine the subcellular localisation of Cd and Zn in mesophyll tissues, protoplasts and vacuoles were isolated from plants exposed to 50 M Cd and Zn hydroponically. Pure protoplasts and vacuoles were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. Of the total Cd and Zn in the mesophyll tissues, 91% and 77%, respectively, were present in the protoplast, and all Cd and 91% Zn in the protoplast were localised in the vacuoles. Furthermore, about 70% and 86% of total Cd and Zn, respectively, in the leaves were extracted in the cell sap, suggesting that most Cd and Zn in the leaves is present in soluble form. These results indicate that internal detoxification of Cd and Zn in Thlaspi caerulescens leaves is achieved by vacuolar compartmentalisation.     

Tissue‐specific isolation of Arabidopsis/plant mitochondria – IMTACT (isolation of mitochondria tagged in specific cell types)

Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue‐specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell‐specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild‐type and mutant Arabidopsis plants we generated a set of OM64–BLRP one‐shot constructs with different selection markers and tissue‐specific promoters.     

In situ localization of glucose and sucrose in dehydrating leaves of Sporobolus stapfianus

Accumulation of soluble carbohydrates during dehydration stress is thought to be a very important mechanism for the acquisition of desiccation tolerance. Despite the proposed importance of soluble carbohydrate accumulation (especially sucrose), nothing is known about the cellular localization of carbohydrates in desiccation-tolerant plants. The present study proposes a novel and selective method for the in situ localization of sucrose and glucose in the desiccation-tolerant plant Sporobolus stapfianus. The detection of sucrose and glucose is based on a series of coupled enzymatic reactions leading to the formation of NADH. Iodonitrotetrazolium (INT) reacts with NADH, thereby providing the red-colored insoluble INT-formazan. Stained tissue sections were immediately visualized using light microscopy. Localization of the respective sugars was site specific. Sucrose was visualized in all leaf cell types during dehydration: vascular bundles, bundle sheath cells, mesophyll cells and epidermal cells. Similarly, glucose was shown to be localized in the same leaf compartments as reported for sucrose. This is the first report that describes sucrose localization in dehydrating leaf tissues of a "resurrection" plant. We conclude that, during dehydration stress, sucrose accumulates in all viable tissues; these results are in agreement with the previously proposed theories about its function as a cellular protectant.     

Structural assessment of the impact of environmental constraints on Arabidopsis thaliana leaf growth: a 3D approach

Light and soil water content affect leaf surface area expansion through modifications in epidermal cell numbers and area, while effects on leaf thickness and mesophyll cell volumes are far less documented. Here, three-dimensional imaging was applied in a study of Arabidopsis thaliana leaf growth to determine leaf thickness and the cellular organization of mesophyll tissues under moderate soil water deficit and two cumulative light conditions. In contrast to surface area, thickness was highly conserved in response to water deficit under both low and high cumulative light regimes. Unlike epidermal and palisade mesophyll tissues, no reductions in cell number were observed in the spongy mesophyll; cells had rather changed in volume and shape. Furthermore, leaf features of a selection of genotypes affected in leaf functioning were analysed. The low-starch mutant pgm had very thick leaves because of unusually large palisade mesophyll cells, together with high levels of photosynthesis and stomatal conductance. By means of an open stomata mutant and a 9-cis-epoxycarotenoid dioxygenase overexpressor, it was shown that stomatal conductance does not necessarily have a major impact on leaf dimensions and cellular organization, pointing to additional mechanisms for the control of CO(2) diffusion under high and low stomatal conductance, respectively.     

Immunolocalization of 1-<Emphasis Type="Italic">O</Emphasis>-sinapoylglucose:malate sinapoyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The serine carboxypeptidase-like protein 1- O-sinapoylglucose:malate sinapoyltransferase (SMT) catalyzes the transfer of the sinapoyl moiety of 1- O-sinapoylglucose to malate in the formation of sinapoylmalate in some members of the Brassicaceae. Rabbit polyclonal monospecific antibodies were raised against the recombinant SMT produced in Escherichia coli from the corresponding Arabidopsis thaliana (L.) Heynh. cDNA. Immunoblot analysis of protein from different Arabidopsis tissues showed that the SMT is produced in all plant organs, except in the seeds and young seedlings. The enzyme was most abundant in older seedlings as well as in rosette leaves and the flowering stem of the plant. Minor amounts were found in the cauline leaves, flower buds and siliques. Traces were detected in the root and flowers. Arabidopsis and transgenic tobacco ( Nicotiana tabacum L.) plants expressing the full-length Arabidopsis SMT containing an N-terminal signal peptide showed apparent molecular masses of the protein of 52-55 kDa. The difference of ca. 8 kDa compared to the recombinant protein produced in E. coli was shown to be due to post-translational N-glycosylation of SMT in plants. Immunofluorescent labeling of Arabidopsis leaf sections localized SMT to the central vacuoles of mesophyll and epidermal cells. Comparable leaf sections of an SMT deletion mutant showed no vacuolar immunofluorescent labeling. We conclude that Arabidopsis SMT is synthesized as a precursor protein that is targeted to the endoplasmic reticulum where the signal peptide is removed. The correct N-terminus of the recombinantly produced SMT protein lacking the signal peptide was confirmed by Edman degradation. The protein is probably glycosylated in the Golgi apparatus from where it is subsequently routed to the vacuole.     

Rumen bacterial degradation of forage cell walls investigated by electron microscopy

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues.     

暗罗属植物叶的比较解剖学研究

利用扫描电镜技术、叶片叶片离析方法和石蜡切片法对暗罗属12种植物叶和形态结构进行了比较研究。结果表明,叶表皮细胞形状、气孔器形态、表皮毛类型、表皮细胞中晶体类型、叶肉中油细胞分布位置、栅栏组织和海绵组织厚度的比值,以及主脉维管组织的结构特征等具有明显的种间差异。可以利用这些叶的解剖特征将暗罗属植物相互区别开来。     

Chalcone synthase and flavonoid products in primary-leaf tissues of rye and maize

During cell and tissue differentiation of developing rye (Secale cereale L.) and maize (Zea mays L.) primary leaves, various flavonoids are synthesized and accumulate in both epidermal and mesophyll tissues. In order to prove either the biosynthetic autonomy of each tissue type and- or intercellular transport of flavonoids, the tissue distributions of chalcone synthase (CHS; EC 2.3.1.74), the key enzyme of the pathway, and of flavonoids have been comparatively investigated. Monoclonal antibodies raised against CHS from rye were used to relate enzyme activity in a particular tissue extract to the corresponding amount of CHS protein. A close correlation was found between CHS activities and amounts of CHS protein during leaf development and in the various tissues. The simultaneous occurrence of CHS in both epidermal layers as well as in the mesophyll correlated with the accumulation of flavonoid products in these tissues, indicating tissue autonomy of flavonoid biosynthesis. These data are in contrast to previous reports (Knogge and Weissenböck, 1986, Planta 167, 196–205) on primary leaves of oat (Avena sativa) where CHS and several subsequent enzymes were located mainly in the mesophyll whereas major flavonoid products accumulated predominantly in both epidermal cell layers, indicating that intertissue transport of flavonoids might occur.     

The Arabidopsis repressor of light signaling SPA1 acts in the phloem to regulate seedling de-etiolation, leaf expansion and flowering time

Plants adjust their growth and development in response to the ambient light environment. These light responses involve systemic signals that coordinate differentiation of different tissues and organs. Here, we have investigated the function of the key repressor of photomorphogenesis SPA1 in different tissues of the plant by expressing GUS-SPA1 under the control of tissue-specific promoters in a spa mutant background. We show that SPA1 expression in the phloem vasculature is sufficient to rescue the spa1 mutant phenotype in dark-grown spa mutant seedlings. Expression of SPA1 in mesophyll, epidermis or root tissues of the seedling, by contrast, has no or only slight effects. In the leaf, SPA1 expression in both the phloem and the mesophyll is required for full complementation of the defect in leaf expansion. SPA1 in phloem and mesophyll tissues affected division and expansion of cells in the epidermal layer, indicating that SPA1 induces non-cell-autonomous responses also in the leaf. Photoperiodic flowering is exclusively controlled by SPA1 expression in the phloem, which is consistent with previous results showing that the direct substrate of the COP1/SPA complex, CONSTANS, also acts in the phloem. Taken together, our results highlight the importance of phloem vascular tissue in coordinating growth and development. Because the SPA1 protein itself is incapable of moving from cell to cell, we suggest that SPA1 regulates the activity of downstream component(s) of light signaling that subsequently act in a non-cell-autonomous manner. SPA1 action in the phloem may also result in mechanical stimuli that affect cell elongation and cell division in other tissues.     

Metabolic engineering of biomass for high energy density: oilseed‐like triacylglycerol yields from plant leaves

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co‐expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild‐type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil‐processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.     

云南秋海棠属植物叶片横切面比较解剖研究

报道30种主产于云南的秋海棠属植物叶片的横切面解剖构造特征。采用常规石蜡切片法切片观察,结果表明:云南秋海棠属植物叶片薄、横切面均为异面叶、呈典型的阴叶结构,叶肉组织虽有栅栏组织和海绵组织的分化,但栅栏组织不发达,占叶肉组织的比例较小。表皮多为单表皮,极稀复表皮,表皮毛均由多细胞组成。气孔集中于下表皮,孔下室或下陷气孔特大、通气组织极发达;角质层形状多样,呈均匀增厚、瘤状和片状突起;叶绿体椭球形、数多、个体大,主要分布于叶肉组织,集中于栅栏组织。解剖构造特征在各分类组内呈现不完全一致性,而在相同茎的形态类型中有些较一致的特征,在不同种间解剖特征各有差别;根状茎和直立茎类型种类的横切面组织结构表现为表皮细胞壁外的角质层薄、栅栏组织与叶肉组织厚度比例较小等弱光照、湿生等适应性较弱的特征。球茎类型的种类表现为角质层较厚、栅栏叶肉组织厚度比例较大等适应略为干燥和较强光照的特征。     

青藏高原草地植物叶解剖特征

运用常规石蜡制片技术对我国青藏高原66种草地植物优势种的叶解剖特征进行研究,并分析了叶解剖特征与海拔、生长季降水及生长季均温之间的关系.结果表明:青藏高原草地植物叶片具有很多适应高寒环境的结构特征,如表皮层厚且表皮细胞大小差异显著,表皮毛等表皮附属物发达,异细胞丰富,通气组织普遍发达等;叶片各组成部分厚度的变异程度不同,其中海绵组织厚度变异最大,其次为上角质层、下表皮层、下角质层、上表皮层、栅栏组织,叶片厚度的变异最小;青藏高原草地植物叶片各组成部分的厚度存在协同进化,上下角质层厚度呈强烈正相关,海绵组织厚度与叶片厚度相关性最强;青藏高原草地植物叶片各组成部分的厚度与海拔、生长季降水、生长季均温3个重要环境变量呈较弱的相关性,总体表现为随海拔升高叶片各组成部分的厚度减小,而随生长季降水和生长季均温的增加叶片厚度增加.