Plant Physiol.-2013-Kellermeier-Natural Variation of Arabidopsis Root Architecture

【PlantPhysiol】Md...文章信息题目：MdWRKY126 modulates malate accumulation in apple fruit by regulating cytosolic malate dehydrogenase (MdMDH5)刊名：Plant Physiology作者：Fengwang Ma, Mingjun Li et al.单位：Northwest A&F University日期：25 January 202201摘要有机酸的含量极大地影响肉质水果的口感和贮藏寿命。

我们目前对苹果（Malus domestica）果实中有机酸积累的分子机制的理解集中于铝激活的苹果酸转运蛋白9/Ma1基因。

在这项研究中，我们使用Ma1的纯合隐性突变体，即Belle de Boskoop“BSKP”和Aifeng“AF”，鉴定了一个独立于Ma1控制水果酸度的候选基因MdWRKY126。

对转基因苹果愈伤组织和果肉和番茄（Solanum lycopersicum）果实的分析表明，MdWRKY126与苹果酸含量基本相关。

MdWRKY126直接与细胞质NAD依赖性苹果酸脱氢酶MdMDH5的启动子结合，并促进其表达，从而提高苹果果实的苹果酸含量。

在MdWRKY126过表达的愈伤组织中，苹果酸相关转运蛋白和质子泵基因的mRNA水平也显著增加，这有助于将积累在细胞质中的苹果酸转运至液泡。

这些发现表明，MdWRKY126调节细胞质中的苹果酸合成代谢，并协调细胞质和液泡之间的运输，以调节苹果酸的积累。

我们的研究为提高我们对调节苹果果实酸度的复杂机制的理解提供了有用的信息。

02技术路线建筑物进入许可关于口罩佩戴校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点校园新冠检测地点03主要结果3.1 候选基因MdWRKY126的鉴定、同源性分析、表达及其蛋白的亚细胞定位在我们之前的研究中，通过酸性（“BSKP”）和非酸性（“AF”）苹果果实的基因表达分析，在转录组数据中发现了77个与苹果果实酸度相关的候选基因。

Breakthrough TechnologiesCorrelative Imaging of Fluorescent Proteins inResin-Embedded Plant Material1Karen Bell,Steve Mitchell,Danae Paultre,Markus Posch,and Karl Oparka*Institute of Molecular Plant Sciences,University of Edinburgh,Edinburgh EH93JR,United Kingdom(K.B., S.M.,D.P.,K.O.);and Light Microscopy Facility,College of Life Sciences,University of Dundee,Dundee DD1 5EH,United Kingdom(M.P.)Fluorescent proteins(FPs)were developed for live-cell imaging and have revolutionized cell biology.However,not all plant tissues are accessible to live imaging using confocal microscopy,necessitating alternative approaches for protein localization.An example is the phloem,a tissue embedded deep within plant organs and sensitive to damage.To facilitate accurate localization of FPs within recalcitrant tissues,we developed a simple method for retaining FPs after resin embedding.This method is based on low-temperaturefixation and dehydration,followed by embedding in London Resin White,and avoids the need for cryosections.We show that a palette of FPs can be localized in plant tissues while retaining good structural cell preservation, and that the polymerized block face can be counterstained with cell wall ing this method we have been able to image greenfluorescent protein-labeled plasmodesmata to a depth of more than40m m beneath the resin ing correlative light and electron microscopy of the phloem,we were able to locate the same FP-labeled sieve elements in semithin and ultrathin sections.Sections were amenable to antibody labeling,and allowed a combination of confocal and superresolution imaging (three-dimensional-structured illumination microscopy)on the same cells.These correlative imaging methods shouldfind several uses in plant cell biology.The localization offluorescent proteins(FPs)in cells and tissues has become one of the major tools in cell bi-ology(Tsien,1998;Shaner et al.,2005).Advances in confocal microscopy have meant that many proteins can be tagged with appropriatefluorescent markers and tracked as they move within and between cells (Chapman et al.,2005).Additional approaches involving photobleaching and photoactivation of FPs have opened up new avenues for exploring protein dynamics and turnover within cells(Lippincott-Schwartz et al.,2003). However,not all cells are amenable to live-cell imaging, which in plants is usually restricted to surface cells such as the leaf epidermis.An example is the phloem.The delicate nature of sieve elements and companion cells, which are under substantial hydrostatic pressure,has made studies of thefine structure of these cells partic-ularly difficult(Knoblauch and van Bel,1998).Despite this,significant advances have been made in imaging the phloem through inventive use of imaging protocols that allow living sieve elements to be observed as they translocate assimilates(for review,see Knoblauch and Oparka,2012).However,determining the precise local-ization of the plethora of proteins located within the sieve element(SE)-companion cell(CC)complex remains a technical challenge.The phloem is the conduit for long-distance movement of macromolecules in plants,including viral genomes.For several viruses,the entry into the SE-CC complex is a crucial step that de-termines the capacity for long-distance movement.Iden-tifying the cell types within the phloem that restrict the movement of some viruses is technically challenging due to the small size of phloem cells and their location deep within plant organs(Nelson and van Bel,1998).The problems associated with imaging proteins in phloem tissues prompted us to explore methods for retaining thefluorescence of tagged proteins within tis-sues not normally amenable to confocal imaging.Previ-ously,we used superresolution imaging techniques on fixed phloem tissues sectioned on a Vibroslice,providing information on the association between a viral movement protein(MP)and plasmodesmata(PD)within the SE-CC complex(Fitzgibbon et al.,2010).However,we wished to explore the same cells using correlative light and electron microscopy(CLEM),necessitating the development of methods that would allow sequential imaging of cells usingfluorescence microscopy and transmission electron microscopy(TEM).To this end,we developed a protocol that retainsfluorescent proteins through aldehydefixa-tion and resin embedding.In the last10years there has been significant interest in imagingfluorescent proteins in semithin sections (for review,see Cortese et al.,2009).Luby-Phelps and colleagues(2003)first described a method for retaining GFPfluorescence afterfixation and resin embedding, but their method has not seen widespread application. The advent of superresolution imaging techniques(for review,see Bell and Oparka,2011)has stimulated considerable interest in thisfield as the retention of1This work was supported by the Biotechnology and Biological Sciences Research Council and the Scottish Universities Life Sciences Alliance.*Corresponding author;e-mail karl.oparka@.The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors()is: Karl Oparka(karl.oparka@)./cgi/doi/10.1104/pp.112.212365fluorescence in thin sections means that cells can be imaged using techniques such as photoactivation light microscopy and stochastic optical reconstruction mi-croscopy,allowing a lateral resolution of less than10 nm to be achieved(Subach et al.,2009;Xu et al.,2012).A number of studies have described CLEM on the same cells(Luby-Phelps et al.,2003;Betzig et al.,2006;Wata-nabe et al.,2011).Advances in thisfield were reviewed recently(Jahn et al.,2012;see contributions in Muller-Reichert and Verkade,2012).For example,Pfeiffer et al. (2003)were able to image SEs and CCs using high-pressure freezing,followed by freeze substitution in ac-etone and resin embedding.They then used thick optical sections of the tissue to locate cells of interest,and these were subsequently imaged using TEM.However,there have been few attempts to retain FPs in resin-embedded plant tissues.Thompson and Wolniak(2008)de-scribed the retention of mCitrine fused to an SE-plasma membrane protein in glycol methacrylate sections.Thefluorescent signal was stable using wide-field microscopy but bleached rapidly under the confocal microscope.To date,cryosections have been the preferred choice for CLEM in mammalian tissues(Watanabe et al.,2011). Recently,Lee et al.(2011)chemicallyfixed Arabidopsis (Arabidopsis thaliana)seedlings,cut50-m m sections,and examined these with a confocal microscope.After con-focal mapping the sections were embedded in resin and thin sectioned.These authors were able to locate the same PD pitfields using confocal and TEM,providing important information on the localization of a novel PD protein.As general rule,cryosectioning is a time-consuming process,and subcellular details may be ob-scured in cryosections because of poor tissue contrast (Watanabe et al.,2011).A major problem with imaging FPs in resin sections has been that GFP and its deriva-tives are quenched by the acidic,oxidizing conditions required forfixation,dehydration,and embedding of delicate specimens(Tsien,1998;Keene et al.,2008).Re-cently,however,Watanabe et al.(2011)explored the retention of FPs in Caenorhabditis elegans cells afterfixa-tion by different aldehydes and embedding media. These authors tested a range of resins and found that Citrine and tandem dimer Eos(tdEos)could be retained in methacrylate plastic sections.This material was dif-ficult to cut thinly(,70nm)compared to epoxy-based resins,but the authors obtained valuable correlative images using stimulated emission depletion microscopy and photoactivation light microscopy followed by low-voltage scanning electron microscopy.Because the retention offluorescent proteins may differ between plant and animal cells,we explored a number of approaches for retainingfluorescent proteins in ing low-temperature conditions(,8°C) duringfixation and dehydration,we could retain strong fluorescence prior to tissue embedding.We also ex-plored different embedding media and found that tis-sue could be effectively polymerized in London Resin (LR)White while retaining sufficientfluorescence for confocal ing water-dipping lenses,we were able to detectfluorescent proteins in optical sections up to40m m below the surface of the block face.Ultrathin sections from the same blocks showed good structural preservation and allowed CLEM.Subsequently,we cut 1-to2-m m sections and examined these using confocal microscopy and three-dimensional-structured illumina-tion microscopy(3D-SIM).Sections could be counter-stained with a number of conventionalfluorophores and antibodies,allowing colocalization studies.These simple methods allow successive imaging of FPs with the light and electron microscope,combining the strengths of both imaging platforms.We believe this approach will have significant utility for tissues that are recalcitrant to conventional confocal imaging.RESULTSFixation and DehydrationBecause a variety of methods have been used to re-tain FPs in semithin sections,we conducted a series of tests on tobacco(Nicotiana tabacum)and Arabidopsis plants expressing different FPs.For these tests we used a transgenic tobacco line in which HDEL:GFP was expressed under the SEO2promoter(Knoblauch and Peters,2010),which is active only in sieve elements.In this line,GFP is targeted to the sieve element reticulum (SER),a specialized form of endoplasmic reticulum that exists as an anastomosing network of tubules and pa-rietal stacked aggregates(Knoblauch and Peters,2010). This line has discrete GFPfluorescence readily visible in freehand sections(Fig.1A).This signal was monitored throughfixation and embedding and allowedfluores-cence levels to be assessed during optimization of the method.Previously we showed that GFPfluorescence, antigenicity,and structural integrity were well pre-served in tissuefixed using a combination of4%para-formaldehyde and0.25%glutaraldehyde(Fitzgibbon et al.,2010).The aim here was to preserve the tissue sufficiently so that it could withstand the rigors of both light and electron microscopy.As expected,we found thatfixation and dehydration at room temperature eliminatedfluorescence before the samples were em-bedded(Keene et al.,2008).However,fixation and dehydration at low temperature(,8°C)successfully retainedfluorescence.We were able to increase the glutaraldehyde concentration as high as2%,while still retaining goodfluorescence preservation.How-ever,we noticed a concomitant increase in the auto-fluorescence of the tissues,particularly the xylem,at this higher glutaraldehyde concentration.However, thefluorescent signal from GFP was easily visible above background(Fig.1B).To limit background auto-fluorescence during processing we included dithiothreitol (DTT)during dehydration and infiltration.When used in combination with low temperature processing,DTT re-duces background autofluorescence(Brown et al.,1989), preserves antigenicity during chemicalfixation(Baskin et al.,1992,1996),and may prevent quenching offluo-rescent proteins(Thompson and Wolniak,2008).Bell et al.Resin EmbeddingWe tested a number of resins including LR White,methacrylate,and Durcupan,a water-miscible resin.Following dehydration,we attempted low-temperature,ultraviolet-,and heat-polymerizing protocols.Material embedded in Durcupan did not section well in our hands.Methacrylate retained fluorescence well after low-temperature/ultraviolet polymerization,but signif-icant tissue collapse was evident (data not shown).Our best results were obtained by polymerizing samples at 50°C in LR White following low-temperature fixation and dehydration.We monitored loss of fluorescence from tissue sections using ImageJ software and found a 27.5%(66,n =11)loss of fluorescence relative to fresh tissue during fixation and embedding.We deemed thisto be an acceptable loss and pursued optimization of subsequent steps.It is likely that plant tissues respond differently to fixatives and embedding media.However,the protocol detailed in the methods section was suitable for most of the tissues we examined.Imaging FPs en Bloc and in Semithin SectionsWe showed previously that plant cell walls can be imaged successfully en bloc following polymerization of tissues in Araldite (Prior et al.,1999).After fixation and embedding in LR White,we imaged the polymer-ized block face using confocal microscopy.In tobacco phloem tissues expressing HDEL:GFP,we could detect fluorescent phloem bundles en bloc at magni ficationsasFigure 1.En bloc imaging of FPs using confocal microscopy.A,Unprocessed,free-hand section of a tobacco petiole expressing pSEO2.HDEL:GFP (shown in green;Knoblauch and Peters,2010).In this construct,GFP highlights the SER but at this mag-nification reveals general fluorescence from phloem bundles.p,Phloem,x,xylem.Scale =600m m.B,Petiole expressing pSEO2.HDEL:GFP imaged in a polymerized block of LR.Scale =600m m.C,An embedded petiole expressing pSEO2.HDEL:GFP imaged with a 633water-dipping lens.The SER is clearly visible at this magnification.Cell walls (blue)were highlighted with calcofluor white,which was added directly to the block face.Scale =40m m.D,A region of the phloem at higher magnification.SEs (se)show conspicuous labeling of the SER,while CCs (cc)show background autofluorescence.Scale =10m m.E–G,Imaging of an Arabidopsis line expressing a viral movement protein fused to GFP (MP17:GFP;Vogel et al.,2007).GFP signal is evident from plasmodesmata (arrow)in mesophyll cells of the leaf.Cell walls were counterstained en bloc with propidium iodide (red).The block was optically sectioned and images captured at the block surface (0m m;E),at 232m m (F),and at 242m m (G)below the block surface.Note that GFP fluorescence from PD is apparent to a depth greater than the penetration of the propidium iodide stain.Scale =50m m.H,En bloc imaging of SEOR1protein (arrows)tagged with YFP (see Froelich et al.,2011)in the phloem of the midvein of an Arabidopsis leaf.Scale =25m m.I,En bloc reconstruction of a viral X-body produced by a PVX vector modified to express GFP fused to its coat protein (CP:GFP;Santa-Cruz et al.,1996).Scale =25m m.J,Nuclei in the hypocotyl of Arabidopsis expressing a histone 2B fused to RFP (H2B:RFP;Federici et al.,2012).Cell walls were counterstained with calcofluor.Scale =25m m.Fluorescent Proteins in Resinlow as53(Fig.1B).We were able to counterstain the cell walls en bloc by adding10m g mL21calcofluor white (Hahne et al.,1983)or1m g mL21propidium iodide (Pighin et al.,2004)directly to the block face as droplets. Using a633lens,we obtained a strong GFP signal from the SER while the calcofluor staining clearly delineated the cell walls(Fig.1C).We found that glutaraldehyde fixation caused a faint background autofluorescence from the cytoplasm,allowing CCs to be identified(Fig.1D). When we used propidium iodide as a wall stain,we found that the cell walls became labeled to a depth of more than30m m into the tissue,allowing deep confocal imaging using water-dipping lenses.In a transgenic line expressing a viral MP fused to GFP(MP17-GFP;Vogel et al.,2007),we were able to imagefluorescent PD in leaf mesophyll cells to a depth of more than40m m into the resin block(Fig.1E–G).At this depth,the propidium io-dide signal had faded significantly,but the MP17-GFP signal remained strong(Fig.1G).SEs in Arabidopsis are extremely small(Mullendore et al.,2010),making SE substructures difficult to detect in semithin ing an Arabidopsis line express-ing yellowfluorescent protein(YFP)fused to the sieve-element occlusion related(SEOR1)protein(Froelich et al., 2011),we were able to image phloem protein bodies within individual sieve elements en bloc(Fig.1H).Next we embedded tobacco leaf petioles infected with a Potato Virus X(PVX)vector in which GFP is fused to the viral coat protein(PVX.CP-GFP;Santa Cruz et al.,1996).In this virus,the GFP forms a virion“overcoat,”allowing the virus to be tracked as it moves.As expected,we found large aggregates of virus particles associated with the viral X-bodies,structures that harbor a range of viral and host components(Tilsner et al.,2012).Using en bloc imaging we could reconstruct individual X-bodies using optical sectioning and reconstruction(Fig.1I).We also embedded an Arabidopsis line expressing a histone2B-redfluorescent protein(RFP)fusion(Federici et al.,2012). Here we were able to image RFP-labeled nuclei in resin sections of the hypocotyl(Fig.1J).CLEMFollowing observation of the block face in the con-focal microscope,we cut ultrathin sections(60nm)for electron microscopy and stained these with uranyl ac-etate and lead citrate.We attempted to image these thin sections in the confocal microscope,prior to heavy-metal staining,but were unable to detect a GFP signal (see also Keene et al.,2008).Ultrathin sections of the phloem expressing HDEL:GFP showed good structural preservation,despite the lack of osmication(Fig.2A). We imaged several phloem bundles in the petiole using TEM and were able to identify the same cells in the block face in the confocal microscope(Fig.2B).In Figure 2C,note that in addition to thefluorescent SER,small vacuoles in the cytoplasm of parenchyma cells can be seen in both the TEM and confocal images.In a number of sections,we were able to identifyfluorescent parietal SER aggregates(Fig.2D)that could also be detected in ultrathin sections with the TEM(Fig.2,E and F).Correlative3D-SIM,Confocal Microscopy,and TEM Most superresolution imaging approaches require that the cells of interest lie close to the coverslip to maximize spatial resolution(Huang et al.,2009;Bell Figure2.CLEM of pSEO2.HDEL:GFP.A,TEM image of an ultrathin section of petiole from a plant expressing pSEO2.HDEL:GFP.The section was poststained with uranyl acetate and lead citrate.Scale=5m m.B,A semithin section acquired imme-diately after the TEM section,imaged with the confocal microscope,showing the samefield of view.Note that small vacuoles inthe cytoplasm can be seen in both the TEM and confocal images(stars in A and B).Scale=5m m.C,Overlay image of A and Bshowing alignment of sieve elements in the confocal and TEM images.D,A Semithin section of the phloem imaged in theconfocal microscope shows conspicuous SER stacks(arrow).Scale=10m m.E,The TEM image of the samefield of view.Thesame SER stack arrowed in D is apparent in E(arrow).Scale=5m m.F,An enlarged image of the SER stack arrowed in E.Scale=1m m.Bell et al.and Oparka,2011).The retention of GFP in semithin sections meets these requirements and allows super-resolution imaging.After confocal imaging of the block face,we cut ultrathin sections and a semithin section from the same region of the ing3D-SIM,we obtained images of sieve plate pores that revealed spa-tial information not present using confocal microscopy or TEM.Figure3,A–D,shows sieve plates imaged se-quentially by confocal laser scanning microscopy (CLSM),TEM,and3D-SIM.Note that each method re-veals different information on the structure of the sieve ing CLSM we could detect,but not resolve, sieve plate pores and the SER associated with them(Fig. 3A).In the thin-section TEM image,we could resolve sieve plate pores and their callose collars,but only par-tial pore transects were encountered due to the section thickness(Fig.3,B and C).In the3D-SIM image,in which we were able to take sequential Z-sections at125-nm spacing,we were able to reconstruct portions of the sieve plate within the thickness of the section(Fig.3D). 3D-SIM resolved the sieve plate pores and revealed distinct cellulose collars that were not apparent in either the TEM or CLSM images(Fig.3D).Ourfixation pro-tocol preserved thefine structure of the SER,which in glancing at sections of sieve plates appeared as afine mesh of interconnected tubules(Fig.3E).Using3D-SIM imaging,we could detect similarfine tubules offluo-rescent SER associated with sieve plates(Fig.3D).These correlative imaging approaches reveal that the three forms of microscopy adopted here(CLSM,3D-SIM,and TEM)are complementary,each revealing important in-formation on subcellular structure.We also examined sections of tissue infected by PVX. CP-GFP using3D-SIM.We were able to imagefine bundles of virusfilaments that could be resolved to about100nm in diameter(Fig.3F).Significantly,we saw very little bleaching of the GFP signal during the mul-tiple acquisitions required to generate3D-SIM images.ImmunofluorescenceLR is compatible withfluorescent antibody labeling, so we were able to achieve triple labeling of sieve el-ements by cutting1-to2-m m sections from the blocks and labeling these with an antibody against callose. Callose is a cell wall constituent found at the neck of PD(Simpson et al.,2009)and sieve plates(Fitzgibbon et al.,2010).Using the confocal microscope,we could detect callose at the lateral sieve plates(Alexa594 secondary antibody)along with the SER(HDEL:GFP; Fig.4A).Figure4B is a3D-SIM image of a sieve plate in transverse orientation.The sieve plate callosewas Figure3.Correlative3D-SIM,confocal microscopy,and TEM of the phloem.A,A semithin section of the phloem froma tobacco petiole expressing pSEO2.HDEL:GFP counterstained with calcofluor white to highlight cell walls and a sieve plate (SP).The sieve plate and SER are visible but not resolved.Scale=5m m.B,The TEM image of the samefield reveals details of thesieve plate and resolves sieve plate pores.Scale=5m m.C,Enlargement of the sieve plate region boxed in B,revealing callosecollars(arrow)around the pores.Scale=1m m.D,A3D-SIM image of the same sieve plate was taken using the section shownin A.The3D-SIM image was reconstructed from20serial Z-sections and,unlike the confocal image,resolves distinct cellulosecollars around the sieve plate pores.The SER is visible at the sieve plate(arrow).Scale=5m m.E,Thefine structure of thetubular SER(arrow)is apparent in a glancing transverse section of a sieve plate imaged using TEM.Scale=1m m.F.3D-SIMimage of PVX X-body(see also en bloc image in Fig.1).3D-SIM resolvesfine viralfilaments at around100nm in diameter (arrow).Scale=5m m.Fluorescent Proteins in Resinimmunolabeled and the cellulose highlighted using calcofluor white.The inset is an enlarged view of two of the pores and shows that the cellulose collars(see Fig.3D)form outside the callose pore linings.Figure 4C shows a confocal image of an immunolabeled sieve tube in longitudinal orientation.We used3D-SIM to image the same sieve tube following staining with calcofluor white(Fig.4D).This revealed the arrange-ments of the SER,callose,and cellulose,respectively, on the sieve plate(Fig.4D,i–iv).DISCUSSIONThefield of correlative microscopy has undergone considerable expansion in recent years(Muller-Reichert and Verkade,2012).Correlative microscopy is the ap-plication of two or more microscopy techniques to the same region of a sample,generating complementary structural and chemical information that would not be possible using a single technique(Jahn et al.,2012).In thisfield,new superresolution imaging instruments have bridged the gap between light and electron microscopy (Watanabe et al.,2011).While confocal microscopy has become the mainstay of modern cell biology,there is a growing need to image the localization of proteins with increasing subcellular accuracy(Betzig et al., 2006).Two distinct types of correlative microscopy approaches have been identified;“combinatorial la-beling,”in which two or more labels are identified using different forms of microscopy(e.g.confocal and TEM)and“noncombinatorial labeling,”in which the label appears in only one type of imaging method but allows identification of the same cells using a second method(Jahn et al.,2012).Generally,noncombinatorial labeling involves faster and simpler sample preparation. The technique we have described here,in which FPs are retained infixed and embedded plant cells,is an exam-ple of a noncombinatorial approach that allows the same FP-containing cells to be identified using TEM.However, our method could be adapted to a combinatorial one if the proteins of interest werefirst labeled with a probe (e.g.fluoronanogold or quantum-dot complexes)that would producefluorescent and electron-dense signals in both confocal microscopy and TEM,respectively.A major goal has been to determine precisely the structures within whichfluorescent proteins reside. The technique we have described here retainsfluo-rescent proteins in resin blocks and semithin sections, and allows imaging of those plant tissues thatare Figure4.Immunodetection of callose in semithin sections of resin-embedded material.A,SER stacks(pSEO2.HDEL:GFP)areseen in tranverse sections of sieve elements.Alexa594-conjugated secondary antibody reveals callose at the sites of lateralsieve areas(red;arrow).Scale=25m m.B,3D-SIM image of a sieve plate in transverse orientation.The sieve plate callose waslabeled with anticallose antibody and visualized using an Alexa594secondary antibody(red).The inset is an enlarged view oftwo of the pores and shows that the cellulose collars form outside the callose pore linings.C,Confocal image of a sieve elementin longitudinal orientation.Callose labeling appears at the sieve plate as well as the lateral areas.Scale=10m m.D,3D-SIMimage of the same sieve plate shown in C.The arrangements of the SER,callose,and cellulose are revealed.SER is shown ingreen(pSEO2.HDEL:GFP;i),callose in red(anticallose antibody and Alexa594secondary;ii),cellulose in blue(calcofluor;iii).The merge of all three channels is shown(iv).Scale=5m m.Bell et al.problematic with conventional live-cellfluorescence microscopy.To achieve CLEM wefirst examined the block face,ahead of cutting a semithin section,fol-lowed by ultrathin sections for TEM.These sections showed good structural preservation whenfixed with a combination of glutaraldehyde and parafor-maldehyde.As in previous studies(Keene et al., 2008),we were unable to retain sufficientfluores-cence in the ultrathin sections to achieve correlative imaging on the same ultrathin section.However, using sequential sectioning we were able to locate the same cells andfluorescently tagged structures using confocal imaging and TEM.The retention of fluorescent proteins in semithin sections also allowed us to use superresolution imaging on the same sec-tions as those used for confocal imaging,extending the range of imaging protocols that can be brought to bear on a single sample.Interestingly,sequential imaging did not simply extend the resolution range but also provided new information on subcellular structure.For example,using3D-SIM we were able to resolve distinct cellulose collars surrounding sieve plate pores.These collars were situated outside the central callose collar but were not visible in either the confocal or TEM images.They may have been gen-erated by the formation of sieve plate callose that,as it expanded,compressed the cellulose microfibrils around the pore.AutofluorescenceIn the material we used here,the FP signal was strong and detected easily above background autofluorescence. With increasing glutaraldehyde concentrations we found that the autofluorescence of cells increased.This was particularly true of the xylem,but other cells showed a degree of cytoplasmicfluorescence.In confocal images this was useful in identifying different cell types in the phloem,such as companion cells(e.g.Fig.1D).It is un-likely that our method will work on cells that show a low and/or diffuse FP signal.Ultimately,the method re-quires a trade off betweenfixation andfluorescence that will depend on the questions being addressed.Variability in LR White pHWatanabe et al.(2011)noted that the pH of batches of LR White varied considerably.Generally,FPs are quenched at low pH(,6;Tsien,1998).We noted also that the pH of LR White batches was variable but obtained good FP retention in plant tissues embedded in a LR White pH range extending from4.6to6.5(data not shown).Higher-pH resin batches are to be preferred because of potential quenching offluorophores.LR White may be buffered to higher pH using ethanolamine (Watanabe et al.,2011).However,this may cause the blocks to become brittle and difficult to section(data not shown).Checking the pH of the resin before attempting FP localization is advisable.CONCLUSIONThe greatest utility of our method is likely to be in the imaging of FP-labeled structures that are difficult to image using conventionalfluorescence microscopy.Such imaging is usually conducted on surface cells,or to a depth that can be accommodated by serial optical imaging.While multiphoton microscopy may extend the depth to which such sectioning is possible(Zipfel et al., 2003),resolution becomes limited.We have shown that by imaging plant tissues en bloc using appropriate counterstains,small structures such as PD(,50nm)can be viewed to a depth of more than40m m in leaf cells. Such deep imaging is helpful when trying to locate structures in the block for subsequent electron micro-scope imaging(Prior et al.,1999)and also permits optical reconstruction of cells without painstaking serial sec-tioning.When required,semithin sections of the tissue can be cut,and these can be stained with conventional fluorophores and antibodies.Despite multiple imaging steps on both confocal and3D-SIM microscopes,the fluorescent proteins we studied retained strongfluores-cence and showed little photobleaching.We have been able to return to the same blocks over a period of several months,and so the method is likely to allow long-term preservation offluorescent proteins in resin when sec-tioning is required at a later date.MATERIALS AND METHODSPlant MaterialTobacco(Nicotiana tabacum)plants expressing pSEO2.HDEL:GFP(Knoblauch and Peters,2010)and Nicotiana benthamiana were grown from seed in a heated glasshouse and used in experiments between30and55d old.Arabidopsis(Arabidopsis thaliana) seedlings expressing SEOR1:YFP(Froelich et al.,2011),MP17:GFP(Vogel et al.,2007), and H2B:RFP(Federici et al.,2012)were germinated and grown on Murashige and Skoog media.Arabidopsis plants were used between3and5d post germination.Fixation and EmbeddingFor tobacco,the petiole was cut and immediately submerged in4%(w/v) formaldehyde(Agar Scientific),2%(w/v)glutaraldehyde(TAAB),50m M PIPES, and1m M CaCl2and then trimmed further underfixative to eliminate any po-tential airlocks.The petioles were allowed to transpire thefixative solution via the xylem for60min at room temperature in an illuminated fume hood(see Fitzgibbon et al.,2010).The petiole was then sectioned transversely into about 2-mm transverse slices using a double-edged razor blade.The sections were then returned to thefixative and incubated for16h on a rolling-bed platform in the dark at8°C.Further tissue processing was done at8°C in the dark unless stated otherwise.The sections were then washed in buffer(50m M PIPES,1m M CaCl2) three times for10min before dehydration in a graded ethanol series(50%[v/v], 70%[v/v],and90%[v/v]twice,each for15min).The ethanol solutions also contained1m M DTT to reduce tissue autofluorescence(Brown et al.,1989).The tissue sections were then infiltrated in medium grade LR(London Resin Com-pany)at1:1,1:2,and1:3ratios of90%ethanol(supplemented by1m M DTT)to resin for45min each before two60-min changes in100%LR.Thefinal em-bedding step was done at ambient temperature.The samples were then poly-merized in gelatin capsules(TAAB)at50°C for24h.In the case of Arabidopsis,the seedlings were processed intact and em-bedded as described above.The only deviation was that the H2B:RFP seedlings werefixed with1%glutaraldehyde to maximize RFPfluorescence.Loss of Tissue FluorescenceTo measure losses in FPfluorescence during the above steps,tissue slices were removed at each stage of thefixation and embedding process and theirFluorescent Proteins in Resin。

PlantPhysiol.德国马普洪堡学者姜亮博⼠利⽤多组学⽅法揭⽰KLU对叶⽚寿命和⼲旱的贡献随着以基因组学，转录组学，代谢组学和蛋⽩质组学为代表的⾼通量组学技术的快速发展与成熟，使得整个⽣命科学研究领域发⽣了翻天覆地的⾰命性变化。

前⾯提到每⼀种技术，都能从⼀定⾓度或层⾯极⼤表征出其⽣物学特征。

然⽽，鉴于⽣物其⾃⾝的多层⾯和复杂性，任何单⼀技术都⽆法有效地捕捉到其全貌。

所以，多组学的联合运⽤更加符合实际需要，事实上也逐渐成为⼀个⾮常有效的⼿段去研究⼀个复杂⽣物学现象。

并且在研究⼈类重⼤疾病，农业⽣产和⽣命科学基础研究领域不断展露头⾓。

本研究，综合运⽤基于RNAseq的转录组和基于GC/LC-MS的代谢组来发掘⼀个催化底物未知的P450氧化酶的⽣物学功能【原⽂链接Multi-omics approach reveals the contribution of KLU toleaf longevity and drought tolerance in Arabidopsis】。

该P450氧化酶被命名为KLU，该基因早在1999年就被报道特异地表达在拟南芥的shoot apical meristem。

Loss of function of KLU 突变体呈现出叶速率加快，侧⽣分枝增多，叶⽚，花器官变⼩。

更为惊奇的是，通过观察KLU表达模式和亚细胞定位，推测出KLU可以产⽣⼀个可以移动的⼩分⼦来调控植物的⽣长发育。

除此外，在拟南芥，⽔稻，⽟⽶，番茄过表达KLU会对其的胚胎发育有着重要的影响，表现为整体果实增⼤并且结实率降低。

尽管KLU对植物的⽣长发育有着重要的影响，但其分⼦机制仍然有待研究。

本⽂利⽤多组学（转录组、初级代谢组和植物激素测定）来探究KLU通过哪些重要的途径来影响植物的⽣长发育。

图1. 整体实验设计思路模式图该⽂的实验思路（见图1）是通过⽐较在野⽣型，突变体及在突变体背景下的过表达转基因株系中的代谢组，转录组差异。

【PlantPhysiol】SP...文章信息题目：SPATULA and ALCATRAZ confer female sterility and fruit cavity via mediating pistil development in cucumber 刊名：Plant Physiology作者：Zhihua Cheng，Xiaolan Zhang et al.单位：China Agricultural University日期：07 April 202201摘要水果的种子在植物有性生殖和人类饮食中起着重要作用。

成功的受精包括将花粉管中的精细胞沿着传输通道（TT）输送到子房内的卵细胞。

果腔是直接影响黄瓜（Cucumis sativus）商业价值的不良性状。

然而，在作物中，果腔的形成和生育力决定的调控基因仍然未知。

在这里，我们描述了黄瓜中的一个基本螺旋环螺旋（bHLH）基因SPATULA（CSPT）及其与ALCATRAZ（CsALC）的冗余和发散功能。

CsSPT转录物在生殖器官中富集。

CsSPT突变导致黄瓜生育能力降低60%，种子仅在果实上部产生。

由于心皮分离，Cspt-Csalc突变体表现出完全丧失生育能力和果腔。

进一步的研究表明，双突变体的柱头向外翻转，柱头同一性有缺陷，异常TT中的细胞外基质含量显著减少，这导致花粉管无延伸路径，胚珠无受精。

生化和转录组分析表明，CsSPT和CsALC在同二聚体和异二聚体中发挥作用，通过介导参与TT 发育、生长素介导的信号传导和黄瓜细胞壁组织的基因，赋予果腔和雌性不育。

02技术路线Cucumber (Cucumis sativus L.) inbred line XTMC (North China type 'Xintaimici’)Gene cloning and structural analysisRT-qPCRIn situ hybridizationSubcellular localizationCucumber transformationMeasurement of fruit physiological indexescanning electron microscopy (SEM) observationPollen viability assaysIn vivo pollen tube aniline blue staining Alcian blue staining of transmitting tracts Transcriptome analysisYeast two-hybrid assayBimolecular fluorescence complementationYeast one-hybrid assay03主要结果3.1 黄瓜的生殖器官中富含CsSPT转录物为了探索与黄瓜种子发生相关的基因，在CuGenDB数据库中搜索SPATULA的同源物（SPT:AT4G36930），因为SPT在拟南芥的育性维持中的作用。

PlantPhysiology综述：植物基因组编辑和脱靶变化的相关性用于靶向基因组编辑的定点核酸酶（SDN）是强大的新工具，可将精确的遗传变化引入植物。

像常规杂交和诱导诱变等传统方法一样，基因组编辑旨在提高作物产量和营养。

下一代测序研究表明，农作物物种的整个基因组通常携带数百万个单核苷酸多态性以及许多拷贝数和结构变异。

自发突变以每代每个位点约10-8至10-9的速率发生，而化学处理或电离辐射引起的变异导致更高的突变率。

图 1 比较不同育种策略导入番茄的每个基因组（单个）的SNP和插入缺失的平均数。

数据代表在S.lycopersicum（Heinz 1706参考基因组）的基因组序列与已用于现代番茄品种育种的其他品种或野生近缘种之间的大约SNP数量。

在SDN中，脱靶更改或编辑是发生在与目标编辑区域具有序列相似性的位点上的意外，非特异性突变。

与自然发生在育种种群中或通过诱变方法引入的SDN变异相比，SDN介导的脱靶变异可以导致少量其他遗传变异。

最近的研究表明，使用计算算法设计基因组编辑试剂可以减轻植物的脱靶编辑。

最后，农作物必须经过强有力的选择，才能通过成熟的多代育种，选择和商业品种开发实践来淘汰异型植物。

图2 来自前20个番茄育种国家的官方发布的突变品种数量，显示了用作育种材料的改良品种(橙条)和突变品种(蓝条)的直接释放。

星号表示欧盟国家。

数据来源：突变品种数据库().在这种情况下，与其他育种实践相比，作物的脱靶编辑不会带来新的安全问题。

已经证明，当前一代的基因组编辑技术对于开发具有消费者和农民利益的新植物品种很有用。

基因组编辑可能会伴随着SDN交付的新发展以及基因组表征的增加而提高编辑特异性，从而进一步改善试剂设计和应用。

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduceprecise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10−8 to 10−9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.版权作品，未经PaperRSS书面授权，严禁转载，违者将被追究法律责任。

《Plant Physiology》（双语）教学教案任课教师：王晓峰教授单位：生命科学学院植物学系授课班级：生科丁颖班、农学丁颖班等Introduction计划学时：2 h一.教学目的了解植物生理学的对象、内容、产生和发展及发展趋势。

二.教学重点植物生理学的内容及发展趋势，植物生理学与分子生物学的关系。

三.教学难点植物生理学的发展趋势四.教学方法采用以多媒体教学法为主。

五.教学用具多媒体硬件支持。

六.教学过程●Introduction of my research work briefly (5 min)●Concept of plant physiology and main contents and chapters of this course (20 min) ●Tasks of plant physiology（20 min）Some examples: Photoperiod, Solution culture, Water culture, Senescence, Ethylene, Tissue culture, Plant growth substance, Photomorphogenesis, Etiolation.●Establishment and development of plant physiology（30 min）In ancient China and western countries→Experimentally/scientifically→J.von Liebig’s work→Modern plant physiology. Establishment and development of plant physiology in China.●Perspectives of plant physiology（10 min）Five problems of human beings : Food, Energy, Environment, Resources, Population ●Summary of the contents of introduction（5 min）Chapter 1 Water Metabolism教学章节：植物对水分的需要、植物细胞对水分的吸收、植物根系对水分的吸收、蒸腾作用、植物体内水分的运输、合理灌溉的生理基础计划学时：3 h一、教学目的通过本章学习，主要了解植物对水分吸收、运输及蒸腾作用的基本原理，认识维持植物水分平衡的重要性，为合理灌溉提供理论基础。

Plant physiologyFrom Wikipedia, the free encyclopediaPlant physiology is a subdiscipline of botany concerned with the functioning, or physiology, of plants. Closely related fields include plant morphology (structure of plants), plant ecology (interactions with the environment), phytochemistry (biochemistry of plants), cell biology, and molecular biology.Fundamental processes such as photosynthesis, respiration, plant nutrition, plant hormone functions, tropisms, nastic movements, photoperiodism, photomorphogenesis, circadian rhythms, environmental stress physiology, seed germination, dormancy and stomata function and transpiration, both part of plant water relations, are studied by plant physiologists.ScopeThe field of plant physiology includes the study of all the internal activities of plants—those chemical and physical processes associated with life as they occur in plants. This includes study at many levels of scale of size and time. At the smallest scale are molecular interactions of photosynthesis and internal diffusion of water, minerals, and nutrients. At the largest scale are the processes of plant development, seasonality, dormancy, and reproductive control. Major subdisciplines of plant physiology include phytochemistry (the study of the biochemistry of plants) and phytopathology (the study of disease in plants). The scope of plant physiology as a discipline may be divided into several major areas of research.First, the study of phytochemistry (plant chemistry) is included within the domain of plant physiology. In order to function and survive, plants produce a wide array of chemical compounds not found in other organisms. Photosynthesis requires a large array of pigments, enzymes, and other compounds to function. Because they cannot move, plants must also defend themselves chemically from herbivores, pathogens and competition from other plants. They do this by producing toxins and foul-tasting or smelling chemicals. Other compounds defend plants against disease, permit survival during drought, and prepare plants for dormancy. While other compounds are used to attract pollinators or herbivores to spread ripe seeds.Secondly, plant physiology includes the study of biological and chemical processes of individual plant cells. Plant cells have a number of features that distinguish them from cells of animals, and which lead to major differences in the way that plant life behaves and responds differently from animal life. For example, plant cells have a cell wall which restricts the shape of plant cells and thereby limits the flexibility and mobility of plants. Plant cells also contain chlorophyll, a chemical compound that interacts with light in a way that enables plants to manufacture their own nutrients rather than consuming other living things as animals do.Thirdly, plant physiology deals with interactions between cells, tissues, and organs within a plant. Different cells and tissues are physically and chemically specialized to perform differentfunctions. Roots and rhizoids function to anchor the plant and acquire minerals in the soil. Leaves function to catch light in order to manufacture nutrients. For both of these organs to remain living, the minerals acquired by the roots must be transported to the leaves and the nutrients manufactured in the leaves must be transported to the roots. Plants have developed a number of means by which this transport may occur, such as vascular tissue, and the functioning of the various modes of transport is studied by plant physiologists.Fourthly, plant physiologists study the ways that plants control or regulate internal functions. Like animals, plants produce chemicals called hormones which are produced in one part of the plant to signal cells in another part of the plant to respond. Many flowering plants bloom at the appropriate time because of light-sensitive compounds that respond to the length of the night, a phenomenon known as photoperiodism. The ripening of fruit and loss of leaves in the winter are controlled in part by the production of the gas ethylene by the plant.Finally, plant physiology includes the study of how plants respond to conditions and variation in the environment, a field known as environmental physiology. Stress from water loss, changes in air chemistry, or crowding by other plants can lead to changes in the way a plant functions. These changes may be affected by genetic, chemical, and physical factors.Biochemistry of plantsThe list of simple elements of which plants are primarily constructed—carbon, oxygen, hydrogen, calcium, phosphorus, etc.—is not different from similar lists for animals, fungi, or even bacteria. The fundamental atomic components of plants are the same as for all life; only the details of the way in which they are assembled differs.Despite this underlying similarity, plants produce a vast array of chemical compounds with unusual properties which they use to cope with their environment. Pigments are used by plants to absorb or detect light, and are extracted by humans for use in dyes. Other plant products may be used for the manufacture of commercially important rubber or biofuel. Perhaps the most celebrated compounds from plants are those with pharmacological activity, such as salicylic acid (aspirin), morphine, and digitalis. Drug companies spend billions of dollars each year researching plant compounds for potential medicinal benefits.Constituent elementsPlants require some nutrients, such as carbon and nitrogen, in large quantities to survive. Such nutrients are termed macronutrients, where the prefix macro- (large) refers to the quantity needed, not the size of the nutrient particles themselves. Other nutrients, called micronutrients, are required only in trace amounts for plants to remain healthy. Such micronutrients are usually absorbed as ions dissolved in water taken from the soil, though carnivorous plants acquire some of their micronutrients from captured prey.The following tables list element nutrients essential to plants. Uses within plants are generalized.Macronutrients. (Necessary in large quantities)Element Form of uptake NotesNitrogen NO3–, NH4+Nucleic acids, proteins, hormones, etc.Oxygen O2 H2O Cellulose, starch, other organic compoundsCarbon CO2Cellulose, starch, other organic compoundsHydrogen H2O Cellulose, starch, other organic compoundsPotassium K+Cofactor in protein synthesis, water balance, etc.Calc ium Ca2+Membrane synthesis and stabilizationMagnesium Mg2+Element essential for chlorophyllPhosphorus H2PO4–Nucleic acids, phospholipids, A TPSulfur SO42–Constituent of proteins and coenzymesMicronutrients. (Necessary in small quantities)Element Form of uptake NotesChlorine Cl-Photosystem II and stomata functionIron Fe2+, Fe3+Chorophyll formationBoron HBO3Crosslinking pectinManganese Mn2+Activity of some enzymesZinc Zn2+Involved in the synthesis of enzymes and chlorophyllCopper Cu+Enzymes for lignin synthesisMolybdenum MoO42-Nitrogen fixation, reduction of nitratesNickel Ni2+Enzymatic cofactor in the metabolism of nitrogen compounds PigmentsAmong the most important molecules for plant function are the pigments. Plant pigments include a variety of different kinds of molecules, including porphyrins, carotenoids, and anthocyanins.All biological pigments selectively absorb certain wavelengths of light while reflecting others. The light that is absorbed may be used by the plant to power chemical reactions, while the reflected wavelengths of light determine the color the pigment will appear to the eye.Chlorophyll is the primary pigment in plants; it is a porphyrin that absorbs red and blue wavelengths of light while reflecting green. It is the presence and relative abundance of chlorophyll that gives plants their green color. All land plants and green algae possess two forms of this pigment: chlorophyll a and chlorophyll b. Kelps, diatoms, and other photosynthetic heterokonts contain chlorophyll c instead of b, while red algae possess only chlorophyll a. All chlorophylls serve as the primary means plants use to intercept light in order to fuel photosynthesis.Carotenoids are red, orange, or yellow tetraterpenoids. They function as accessory pigments in plants, helping to fuel photosynthesis by gathering wavelengths of light not readily absorbed by chlorophyll. The most familiar carotenoids are carotene (an orange pigment found in carrots), lutein (a yellow pigment found in fruits and vegetables), and lycopene (the red pigment responsible for the color of tomatoes). Carotenoids have been shown to act as antioxidants and to promote healthy eyesight in humans.Anthocyanins (literally "flower blue") are water-soluble flavonoid pigments that appear red to blue, according to pH. They occur in all tissues of higher plants, providing color in leaves, stems, roots, flowers, and fruits, though not always in sufficient quantities to be noticeable. Anthocyanins are most visible in the petals of flowers, where they may make up as much as 30% of the dry weight of the tissue. They are also responsible for the purple color seen on the underside of tropical shade plants such as Tradescantia zebrina; in these plants, the anthocyanin catches light that has passed through the leaf and reflects it back towards regions bearing chlorophyll, in order to maximize the use of available light.Betalains are red or yellow pigments. Like anthocyanins they are water-soluble, but unlike anthocyanins they are indole-derived compounds synthesized from tyrosine. This class of pigments is found only in the Caryophyllales (including cactus and amaranth), and neverco-occur in plants with anthocyanins. Betalains are responsible for the deep red color of beets, and are used commercially as food-coloring agents. Plant physiologists are uncertain of the function that betalains have in plants which possess them, but there is some preliminary evidence that they may have fungicidal properties.Signals and regulatorsPlants produce hormones and other growth regulators which act to signal a physiological response in their tissues. They also produce compounds such as phytochrome that are sensitive to light and which serve to trigger growth or development in response to environmental signals.Plant hormonesPlant hormones, known as plant growth regulators (PGRs) or phytohormones, are chemicals that regulate a plant's growth. According to a standard animal definition, hormones are signal molecules produced at specific locations, that occur in very low concentrations, and cause altered processes in target cells at other locations. Unlike animals, plants lack specifichormone-producing tissues or organs. Plant hormones are often not transported to other parts of the plant and production is not limited to specific locations.Plant hormones are chemicals that in small amounts promote and influence the growth, development and differentiation of cells and tissues. Hormones are vital to plant growth; affecting processes in plants from flowering to seed development, dormancy, and germination. They regulate which tissues grow upwards and which grow downwards, leaf formation and stem growth, fruit development and ripening, as well as leaf abscission and even plant death.The most important plant hormones are abscissic acid (ABA), auxins, ethylene, gibberellins, and cytokinins, though there are many other substances that serve to regulate plant physiology. PhotomorphogenesisWhile most people know that light is important for photosynthesis in plants, few realize that plant sensitivity to light plays a role in the control of plant structural development (morphogenesis). The use of light to control structural development is called photomorphogenesis, and is dependent upon the presence of specialized photoreceptors, which are chemical pigments capable of absorbing specific wavelengths of light.Plants use four kinds of photoreceptors: phytochrome, cryptochrome, a UV-B photoreceptor, and protochlorophyllide a. The first two of these, phytochrome and cryptochrome, are photoreceptor proteins, complex molecular structures formed by joining a protein with a light-sensitive pigment. Cryptochrome is also known as the UV-A photoreceptor, because it absorbs ultraviolet light in the long wave "A" region. The UV-B receptor is one or more compounds that have yet to be identified with certainty, though some evidence suggests carotene or riboflavin as candidates. Protochlorophyllide a, as its name suggests, is a chemical precursor of chlorophyll.The most studied of the photoreceptors in plants is phytochrome. It is sensitive to light in the red and far-red region of the visible spectrum. Many flowering plants use it to regulate the time of flowering based on the length of day and night (photoperiodism) and to set circadian rhythms. It also regulates other responses including the germination of seeds, elongation of seedlings, the size, shape and number of leaves, the synthesis of chlorophyll, and the straightening of the epicotyl or hypocotyl hook of dicot seedlings.PhotoperiodismMany flowering plants use the pigment phytochrome to sense seasonal changes in day length, which they take as signals to flower. This sensitivity to day length is termed photoperiodism. Broadly speaking, flowering plants can be classified as long day plants, short day plants, or day neutral plants, depending on their particular response to changes in day length. Long day plants require a certain minimum length of daylight to initiate flowering, so these plants flower in the spring or summer. Conversely, short day plants will flower when the length of daylight falls below a certain critical level. Day neutral plants do not initiate flowering based on photoperiodism, though some may use temperature sensitivity (vernalization) instead.Although a short day plant cannot flower during the long days of summer, it is not actually the period of light exposure that limits flowering. Rather, a short day plant requires a minimal length of uninterrupted darkness in each 24 hour period (a short daylength) before floral development can begin. It has been determined experimentally that a short day plant (long night) will not flower if a flash of phytochrome activating light is used on the plant during the night.Plants make use of the phytochrome system to sense day length or photoperiod. This fact is utilized by florists and greenhouse gardeners to control and even induce flowering out of season, such as the Poinsettia.Environmental physiologyParadoxically, the subdiscipline of environmental physiology is on the one hand a recent field of study in plant ecology and on the other hand one of the oldest. Environmental physiology is the preferred name of the subdiscipline among plant physiologists, but it goes by a number of other names in the applied sciences. It is roughly synonymous with ecophysiology, crop ecology, horticulture and agronomy. The particular name applied to the subdiscipline is specific to the viewpoint and goals of research. Whatever name is applied, it deals with the ways in which plants respond to their environment and so overlaps with the field of ecology. Environmental physiologists examine plant response to physical factors such as radiation (including light and ultraviolet radiation), temperature, fire, and wind. Of particular importance are water relations (which can be measured with the Pressure bomb) and the stress of drought or inundation, exchange of gases with the atmosphere, as well as the cycling of nutrients such as nitrogen and carbon.Environmental physiologists also examine plant response to biological factors. This includes not only negative interactions, such as competition, herbivory, disease and parasitism, but also positive interactions, such as mutualism and pollination.Tropisms and nastic movementsPlants may respond both to directional and nondirectional stimuli. A response to a directional stimulus, such as gravity or sunlight, is called a tropism. A response to a nondirectional stimulus, such as temperature or humidity, is a nastic movement.Tropisms in plants are the result of differential cell growth, in which the cells on one side of the plant elongate more than those on the other side, causing the part to bend toward the side with less growth. Among the common tropisms seen in plants is phototropism, the bending of the plant toward a source of light. Phototropism allows the plant to maximize light exposure in plants which require additional light for photosynthesis, or to minimize it in plants subjected to intense light and heat. Geotropism allows the roots of a plant to determine the direction of gravity and grow downwards. Tropisms generally result from an interaction between the environment and production of one or more plant hormones.In contrast to tropisms, nastic movements result from changes in turgor pressure within plant tissues, and may occur rapidly. A familiar example is thigmonasty (response to touch) in the Venus fly trap, a carnivorous plant. The traps consist of modified leaf blades which bear sensitive trigger hairs. When the hairs are touched by an insect or other animal, the leaf folds shut. This mechanism allows the plant to trap and digest small insects for additional nutrients. Although the trap is rapidly shut by changes in internal cell pressures, the leaf must grow slowly in order to reset for a second opportunity to trap insects.Plant diseaseEconomically, one of the most important areas of research in environmental physiology is that of phytopathology, the study of diseases in plants and the manner in which plants resist or cope with infection. Plant are susceptible to the same kinds of disease organisms as animals, including viruses, bacteria, and fungi, as well as physical invasion by insects and roundworms.Because the biology of plants differs from animals, their symptoms and responses are quite different. In some cases, a plant can simply shed infected leaves or flowers to prevent to spread of disease, in a process called abscission. Most animals do not have this option as a means of controlling disease. Plant diseases organisms themselves also differ from those causing disease in animals because plants cannot usually spread infection through casual physical contact. Plant pathogens tend to spread via spores or are carried by animal vectors.One of the most important advances in the control of plant disease was the discovery of Bordeaux mixture in the nineteenth century. The mixture is the first known fungicide and is a combination of copper sulfate and lime. Application of the mixture served to inhibit the growth of downy mildew that threatened to seriously damage the French wine industry.HistoryEarly historySir Francis Bacon published one of the first plant physiology experiments in 1627 in the book, Sylva Sylvarum. Bacon grew several terrestrial plants, including a rose, in water and concluded that soil was only needed to keep the plant upright. Jan Baptist van Helmont published what is considered the first quantitative experiment in plant physiology in 1648. He grew a willow tree for five years in a pot containing 200 pounds of oven-dry soil. The soil lost just two ounces of dry weight and van Helmont concluded that plants get all their weight from water, not soil. In 1699, John Woodward published experiments on growth of spearmint in different sources of water. He found that plants grew much better in water with soil added than in distilled water.Stephen Hales is considered the Father of Plant Physiology for the many experiments in the 1727 book; though Julius von Sachs unified the pieces of plant physiology and put them together as a discipline. His Lehrbuch der Botanik was the plant physiology bible of its time.Researchers discovered in the 1800s that plants absorb essential mineral nutrients as inorganic ions in water. In natural conditions, soil acts as a mineral nutrient reservoir but the soil itself is not essential to plant growth. When the mineral nutrients in the soil are dissolved in water, plant roots absorb nutrients readily, soil is no longer required for the plant to thrive. This observation is the basis for hydroponics, the growing of plants in a water solution rather than soil, which has become a standard technique in biological research, teaching lab exercises, crop production and as a hobby.Current researchOne of the leading journals in the field is Plant Physiology, started in 1926. All its back issues are available online for free. Many other journals often carry plant physiology articles, including Physiologia Plantarum, Journal of Experimental Botany, American Journal of Botany, Annals of Botany, Journal of Plant Nutrition and Proceedings of the National Academy of Sciences. Economic applicationsFood productionIn horticulture and agriculture along with food science, plant physiology is an important topic relating to fruits, vegetables, and other consumable parts of plants. Topics studied include: climatic requirements, fruit drop, nutrition, ripening, fruit set. The production of food crops also hinges on the study of plant physiology covering such topics as Optimal planting and harvesting times and post harvest storage of plant products for human consumption and the production of secondary products like drugs and cosmetics.。

21ChapterCytokinins:Regulators of Cell DivisionTHE CYTOKININS WERE DISCOVERED in the search for factors thatstimulate plant cells to divide (i.e., undergo cytokinesis). Since their dis-covery, cytokinins have been shown to have effects on many other phys-iological and developmental processes, including leaf senescence, nutri-ent mobilization, apical dominance, the formation and activity of shootapical meristems, floral development, the breaking of bud dormancy,and seed germination. Cytokinins also appear to mediate many aspectsof light-regulated development, including chloroplast differentiation,the development of autotrophic metabolism, and leaf and cotyledonexpansion.Although cytokinins regulate many cellular processes, the control ofcell division is central in plant growth and development and is consid-ered diagnostic for this class of plant growth regulators. For these rea-sons we will preface our discussion of cytokinin function with a briefconsideration of the roles of cell division in normal development,wounding, gall formation, and tissue culture.Later in the chapter we will examine the regulation of plant cell pro-liferation by cytokinins. Then we will turn to cytokinin functions notdirectly related to cell division: chloroplast differentiation, the preven-tion of leaf senescence, and nutrient mobilization. Finally, we will con-sider the molecular mechanisms underlying cytokinin perception andsignaling.CELL DIVISION AND PLANT DEVELOPMENTPlant cells form as the result of cell divisions in a primary or secondarymeristem. Newly formed plant cells typically enlarge and differentiate,but once they have assumed their function—whether transport, pho-tosynthesis, support, storage, or protection—usually they do not divideagain during the life of the plant. In this respect they appear to be sim-ilar to animal cells, which are considered to be terminally differentiated.However, this similarity to the behavior of animal cells is only super-ficial. Almost every type of plant cell that retains its nucleus at maturityhas been shown to be capable of dividing. This property comes into play during such processes as wound healing and leaf abscission.Differentiated Plant Cells Can Resume Division Under some circumstances, mature, differentiated plant cells may resume cell division in the intact plant. In many species, mature cells of the cortex and/or phloem resume division to form secondary meristems, such as the vascular cambium or the cork cambium. The abscission zone at the base of a leaf petiole is a region where mature parenchyma cells begin to divide again after a period of mitotic inactiv-ity, forming a layer of cells with relatively weak cell walls where abscission can occur (see Chapter 22).Wounding of plant tissues induces cell divisions at the wound site. Even highly specialized cells, such as phloem fibers and guard cells, may be stimulated by wounding to divide at least once. Wound-induced mitotic activity typi-cally is self-limiting; after a few divisions the derivative cells stop dividing and redifferentiate. However, when the soil-dwelling bacterium Agrobacterium tumefaciens invades a wound, it can cause the neoplastic (tumor-forming) disease known as crown gall. This phenomenon is dramatic natural evidence of the mitotic potential of mature plant cells.Without Agrobacterium infection, the wound-induced cell division would subside after a few days and some of the new cells would differentiate as a protective layer of cork cells or vascular tissue. However, Agrobacterium changes the character of the cells that divide in response to the wound, making them tumorlike. They do not stop dividing; rather they continue to divide throughout the life of the plant to produce an unorganized mass of tumorlike tissue called a gall (Figure 21.1).We will have more to say about this important disease later in this chapter.Diffusible Factors May Control Cell DivisionThe considerations addressed in the previous section sug-gest that mature plant cells stop dividing because they no longer receive a particular signal, possibly a hormone, that is necessary for the initiation of cell division. The idea that cell division may be initiated by a diffusible factor origi-nated with the Austrian plant physiologist G. Haberlandt, who, in about 1913, demonstrated that vascular tissue con-tains a water-soluble substance or substances that will stim-ulate the division of wounded potato tuber tissue. The effort to determine the nature of this factor (or factors) led to the discovery of the cytokinins in the 1950s.Plant Tissues and Organs Can Be Cultured Biologists have long been intrigued by the possibility of growing organs, tissues, and cells in culture on a simple nutrient medium, in the same way that microorganisms can be cultured in test tubes or on petri dishes. In the 1930s, Philip White demonstrated that tomato roots can be grown indefinitely in a simple nutrient medium containing only sucrose, mineral salts, and a few vitamins, with no added hormones (White 1934).In contrast to roots, isolated stem tissues exhibit very lit-tle growth in culture without added hormones in the medium. Even if auxin is added, only limited growth may occur, and usually this growth is not sustained. Frequently this auxin-induced growth is due to cell enlargement only. The shoots of most plants cannot grow on a simple medium lacking hormones, even if the cultured stem tis-sue contains apical or lateral meristems, until adventitious roots form. Once the stem tissue has rooted, shoot growth resumes, but now as an integrated, whole plant.These observations indicate that there is a difference in the regulation of cell division in root and shoot meristems. They also suggest that some root-derived factor(s) may reg-ulate growth in the shoot.Crown gall stem tissue is an exception to these general-izations. After a gall has formed on a plant, heating the plant to 42°C will kill the bacterium that induced gall for-mation. The plant will survive the heat treatment, and its gall tissue will continue to grow as a bacteria-free tumor (Braun 1958).Tissues removed from these bacteria-free tumors grow on simple, chemically defined culture media that would not support the proliferation of normal stem tissue of the same species. However, these stem-derived tissues are not organized. Instead they grow as a mass of disorganized, relatively undifferentiated cells called callus tissue.Callus tissue sometimes forms naturally in response to wounding, or in graft unions where stems of two different plants are joined. Crown gall tumors are a specific type of callus, whether they are growing attached to the plant or in culture. The finding that crown gall callus tissue can be cultured demonstrated that cells derived from stem tissues are capable of proliferating in culture and that contact with494Chapter21FIGURE 21.1 Tumor that formed on a tomato stem infected with the crown gall bacterium, Agrobacterium tumefaciens.Two months before this photo was taken the stem was wounded and inoculated with a virulent strain of the crown gall bac-terium. (From Aloni et al. 1998, courtesy of R. Aloni.)the bacteria may cause the stem cells to produce cell divi-sion –stimulating factors.THE DISCOVERY,IDENTIFICATION,AND PROPERTIES OF CYTOKININSA great many substances were tested in an effort to initiate and sustain the proliferation of normal stem tissues in cul-ture. Materials ranging from yeast extract to tomato juice were found to have a positive effect, at least with some tis-sues. However, culture growth was stimulated most dra-matically when the liquid endosperm of coconut, also known as coconut milk, was added to the culture medium. Philip White ’s nutrient medium, supplemented with an auxin and 10 to 20% coconut milk, will support the con-tinued cell division of mature, differentiated cells from a wide variety of tissues and species, leading to the forma-tion of callus tissue (Caplin and Steward 1948). This find-ing indicated that coconut milk contains a substance or substances that stimulate mature cells to enter and remain in the cell division cycle.Eventually coconut milk was shown to contain the cytokinin zeatin , but this finding was not obtained until several years after the discovery of the cytokinins (Letham 1974). The first cytokinin to be discovered was the synthetic analog kinetin.Kinetin Was Discovered as a Breakdown Product of DNAIn the 1940s and 1950s, Folke Skoog and coworkers at the University of Wisconsin tested many substances for their ability to initiate and sustain the proliferation of cultured tobacco pith tissue. They had observed that the nucleic acid base adenine had a slight promotive effect, so they tested the possibility that nucleic acids would stimulate division in this tissue. Surprisingly , autoclaved herring sperm DNA had a powerful cell division –promoting effect.After much work, a small molecule was identified from the autoclaved DNA and named kinetin . It was shown to be an adenine (or aminopurine) derivative, 6-furfury-In the presence of an auxin, kinetin would stimulate tobacco pith parenchyma tissue to proliferate in culture. No kinetin-induced cell division occurs without auxin in the culture medium. (For more details, see Web Topic 21.1.)Kinetin is not a naturally occurring plant growth regu-lator, and it does not occur as a base in the DNA of any species. It is a by-product of the heat-induced degradation of DNA, in which the deoxyribose sugar of adenosine is converted to a furfuryl ring and shifted from the 9 position to the 6 position on the adenine ring.The discovery of kinetin was important because it demon-strated that cell division could be induced by a simple chem-ical substance. Of greater importance, the discovery of kinetin suggested that naturally occurring molecules with structures similar to that of kinetin regulate cell division activity within the plant. This hypothesis proved to be correct.Zeatin Is the Most Abundant Natural CytokininSeveral years after the discovery of kinetin, extracts of the immature endosperm of corn (Zea mays ) were found to contain a substance that has the same biological effect as kinetin. This substance stimulates mature plant cells to divide when added to a culture medium along with an auxin. Letham (1973) isolated the molecule responsible for this activity and identified it as trans -6-(4-hydroxy-3-methylbut-2-enylamino)purine, which he called zeatin :The molecular structure of zeatin is similar to that of kinetin. Both molecules are adenine or aminopurine derivatives. Although they have different side chains, in both cases the side chain is attached to the 6 nitrogen of the aminopurine. Because the side chain of zeatin has a double bond, it can exist in either the cis or the trans con-figuration.In higher plants, zeatin occurs in both the cis and the trans configurations, and these forms can be interconverted by an enzyme known as zeatin isomerase . Although the trans form of zeatin is much more active in biological assays, the cis form may also play important roles, as suggested by the fact that it has been found in high levels in a number of plant species and particular tissues. A gene encoding a glu-cosyl transferase enzyme specific to cis -zeatin has recently been cloned, which further supports a biological role for this isoform of zeatin.Since its discovery in immature maize endosperm,zeatin has been found in many plants and in some bacte-ria. It is the most prevalent cytokinin in higher plants, but other substituted aminopurines that are active as cytokinins have been isolated from many plant and bac-Cytokinins:Regulators of Cell Division495terial species. These aminopurines differ from zeatin in the nature of the side chain attached to the 6 nitrogen or in the attachment of a side chain to carbon 2:In addition, these cytokinins can be present in the plant as a riboside(in which a ribose sugar is attached to the 9 nitrogen of the purine ring), a ribotide(in which the ribose sugar moiety contains a phosphate group), or a glycoside (in which a sugar molecule is attached to the 3, 7, or 9 nitro-gen of the purine ring, or to the oxygen of the zeatin or dihydrozeatin side chain) (see Web Topic 21.2).Some Synthetic Compounds Can Mimic or Antagonize Cytokinin ActionCytokinins are defined as compounds that have biological activities similar to those of trans-zeatin. These activities include the ability to do the following:•Induce cell division in callus cells in the presence of an auxin•Promote bud or root formation from callus cultures when in the appropriate molar ratios to auxin •Delay senescence of leaves•Promote expansion of dicot cotyledonsMany chemical compounds have been synthesized and tested for cytokinin activity. Analysis of these compounds provides insight into the structural requirements for activ-ity. Nearly all compounds active as cytokinins are N6-sub-and all the naturally occurring cytokinins are aminopurinederivatives. There are also synthetic cytokinin compoundsthat have not been identified in plants, most notable ofwhich are the diphenylurea-type cytokinins, such as thidi-azuron, which is used commercially as a defoliant and anherbicide:In the course of determining the structural requirementsfor cytokinin activity, investigators found that some mole-cules act as cytokinin antagonists:These molecules are able to block the action of cytokinins,and their effects may be overcome by the addition of morecytokinin. Naturally occurring molecules with cytokininactivity may be detected and identified by a combinationof physical methods and bioassays(see Web Topic 21.3).Cytokinins Occur in Both Free and Bound FormsHormonal cytokinins are present as free molecules (notcovalently attached to any macromolecule) in plants andcertain bacteria. Free cytokinins have been found in a widespectrum of angiosperms and probably are universal inthis group of plants. They have also been found in algae,diatoms, mosses, ferns, and conifers.The regulatory role of cytokinins has been demonstratedonly in angiosperms, conifers, and mosses, but they mayfunction to regulate the growth, development, and metab-olism of all plants. Usually zeatin is the most abundant nat-urally occurring free cytokinin, but dihydrozeatin(DZ) andisopentenyl adenine(iP) also are commonly found in higherplants and bacteria. Numerous derivatives of these threecytokinins have been identified in plant extracts (see thestructures illustrated in Figure 21.6).Transfer RNA(tRNA) contains not only the fournucleotides used to construct all other forms of RNA, butalso some unusual nucleotides in which the base has beenmodified. Some of these “hypermodified” bases act ascytokinins when the tRNA is hydrolyzed and tested in oneof the cytokinin bioassays. Some plant tRNAs contain cis-496Chapter21zeatin as a hypermodified base. However, cytokinins are not confined to plant tRNAs. They are part of certain tRNAs from all organisms, from bacteria to humans. (For details, see Web Topic 21.4.)The Hormonally Active Cytokinin Is the Free Base It has been difficult to determine which species of cytokinin represents the active form of the hormone, but the recent identification of the cytokinin receptor CRE1 has allowed this question to be addressed. The relevant experiments have shown that the free-base form of trans-zeatin, but not its ribo-side or ribotide derivatives, binds directly to CRE1, indicat-ing that the free base is the active form (Yamada et al. 2001).Although the free-base form of trans-zeatin is thought to be the hormonally active cytokinin, some other compounds have cytokinin activity, either because they are readily con-verted to zeatin, dihydrozeatin, or isopentenyl adenine, or because they release these compounds from other mole-cules, such as cytokinin glucosides. For example, tobacco cells in culture do not grow unless cytokinin ribosides sup-plied in the culture medium are converted to the free base.In another example, excised radish cotyledons grow when they are cultured in a solution containing the cytokinin base benzyladenine (BA, an N6-substituted aminopurine cytokinin). The cultured cotyledons readily take up the hormone and convert it to various BA gluco-sides, BA ribonucleoside, and BA ribonucleotide. When the cotyledons are transferred back to a medium lacking a cytokinin, their growth rate declines, as do the concentra-tions of BA, BA ribonucleoside, and BA ribonucleotide in the tissues. However, the level of the BA glucosides remains constant. This finding suggests that the glucosides cannot be the active form of the hormone.Some Plant Pathogenic Bacteria,Insects,and Nematodes Secrete Free CytokininsSome bacteria and fungi are intimately associated with higher plants. Many of these microorganisms produce and secrete substantial amounts of cytokinins and/or cause the plant cells to synthesize plant hormones, including cytokinins (Akiyoshi et al. 1987). The cytokinins produced by microorganisms include trans-zeatin, [9R]iP, cis-zeatin, and their ribosides (Figure 21.2). Infection of plant tissues with these microorganisms can induce the tissues to divide and, in some cases, to form special structures, such as myc-orrhizae, in which the microorganism can reside in a mutu-alistic relationship with the plant.In addition to the crown gall bacterium, Agrobacterium tumefaciens, other pathogenic bacteria may stimulate plant cells to divide. For example, Corynebacterium fascians is a major cause of the growth abnormality known as witches’-broom(Figure 21.3). The shoots of plants infected by C. fas-cians resemble an old-fashioned straw broom because the lateral buds, which normally remain dormant, are stimu-lated by the bacterial cytokinin to grow (Hamilton and Lowe 1972).Cytokinins:Regulators of Cell Division497FIGURE 21.2Structures of ribosylzeatin and N6-(∆2-isopen-tenyl)adenosine ([9R]iP).FIGURE 21.3Witches’ broom on balsam fir (Abies balsamea). (Photo © Gregory K. Scott/Photo Researchers, Inc.)Infection with a close relative of the crown gall organ-ism, Agrobacterium rhizogenes, causes masses of roots instead of callus tissue to develop from the site of infec-tion. A. rhizogenes is able to modify cytokinin metabolism in infected plant tissues through a mechanism that will be described later in this chapter.Certain insects secrete cytokinins, which may play a role in the formation of galls utilized by these insects as feeding sites. Root-knot nematodes also produce cytokinins, which may be involved in manipulating host development to produce the giant cells from which the nematode feeds (Elzen 1983). BIOSYNTHESIS,METABOLISM,ANDTRANSPORT OF CYTOKININSThe side chains of naturally occurring cytokinins are chemically related to rubber, carotenoid pigments, the plant hormones gibberellin and abscisic acid, and some of the plant defense compounds known as phytoalexins. All of these compounds are constructed, at least in part, from isoprene units (see Chapter 13).Isoprene is similar in structure to the side chains of zeatin and iP(see the structures illustrated in Figure 21.6). These cytokinin side chains are synthesized from an iso-prene derivative. Large molecules of rubber and the carotenoids are constructed by the polymerization of many isoprene units; cytokinins contain just one of these units. The precursor(s) for the formation of these isoprene structures are either mevalonic acid or pyruvate plus 3-phosphoglycerate, depending on which pathway is involved (see Chapter 13). These precursors are converted to the biological isoprene unit dimethylallyl diphosphate (DMAPP).Crown Gall Cells Have Acquired a Gene for Cytokinin SynthesisBacteria-free tissues from crown gall tumors proliferate in culture without the addition of any hormones to the cul-ture medium. Crown gall tissues contain substantial amounts of both auxin and free cytokinins. Furthermore, when radioactively labeled adenine is fed to periwinkle (Vinca rosea) crown gall tissues, it is incorporated into both zeatin and zeatin riboside, demonstrating that gall tissues contain the cytokinin biosynthetic pathway. Control stem tissue, which has not been transformed by Agrobacterium, does not incorporate labeled adenine into cytokinins.During infection by Agrobacterium tumefaciens, plant cells incorporate bacterial DNA into their chromosomes. The virulent strains of Agrobacterium contain a large plas-mid known as the Ti plasmid.Plasmids are circular pieces of extrachromosomal DNA that are not essential for the life of the bacterium. However, plasmids frequently con-tain genes that enhance the ability of the bacterium to sur-vive in special environments.A small portion of the Ti plasmid, known as the T-DNA, is incorporated into the nuclear DNA of the host plant cell (Figure 21.4) (Chilton et al. 1977). T-DNA carries genes necessary for the biosynthesis of trans-zeatin and auxin, as well as a member of a class of unusual nitrogen-containing compounds called opines(Figure 21.5). Opines are not synthesized by plants except after crown gall trans-formation.The T-DNA gene involved in cytokinin biosynthesis—known as the ipt1gene—encodes an i so p entenyl t rans-ferase (IPT)enzyme that transfers the isopentenyl group from DMAPP to AMP(adenosine monophosphate) to form isopentenyl adenine ribotide (Figure 21.6) (Akiyoshi et al. 1984; Barry et al. 1984). The ipt gene has been called the tmr locus because, when inactivated by mutation, it results in “rooty” tumors. Isopentenyl adenine ribotide can be con-verted to the active cytokinins isopentenyl adenine,trans-zeatin, and dihydrozeatin by endogenous enzymes in plant cells. This conversion route is similar to the pathway for cytokinin synthesis that has been postulated for normal tis-sue (see Figure 21.6).The T-DNA also contains two genes encoding enzymes that convert tryptophan to the auxin indole-3-acetic acid (IAA). This pathway of auxin biosynthesis differs from the one in nontransformed cells and involves indoleacetamide as an intermediate (see Figure 19.6). The ipt gene and the two auxin biosynthetic genes of T-DNA are phyto-onco-genes, since they can induce tumors in plants (see Web Topic 21.5).Because their promoters are plant eukaryotic promoters, none of the T-DNA genes are expressed in the bacterium; rather they are transcribed after they are inserted into the plant chromosomes. Transcription of the genes leads to synthesis of the enzymes they encode, resulting in the pro-duction of zeatin, auxin, and an opine. The bacterium can utilize the opine as a nitrogen source, but cells of higher plants cannot. Thus, by transforming the plant cells, the bacterium provides itself with an expanding environment (the gall tissue) in which the host cells are directed to pro-duce a substance (the opine) that only the bacterium can utilize for its nutrition (Bomhoff et al. 1976).An important difference between the control of cytokinin biosynthesis in crown gall tissues and in normal tissues is that the T-DNA genes for cytokinin synthesis are expressed in all infected cells, even those in which the native plant genes for biosynthesis of the hormone are nor-mally repressed.IPT Catalyzes the First Step in Cytokinin BiosynthesisThe first committed step in cytokinin biosynthesis is the transfer of the isopentenyl group of dimethylallyl diphos-498Chapter211Bacterial genes, unlike plant genes, are written in lower-case italics.phate (DMAPP) to an adenosine moiety. An enzyme that catalyzes such an activity was first identified in the cellu-lar slime mold Dictyostelium discoideum, and subsequently the ipt gene from Agrobacterium was found to encode such an enzyme. In both cases, DMAPP and AMP are converted to isopentenyladenosine-5′-monophosphate (iPMP).As noted earlier, cytokinins are also present in the tRNAs of most cells, including plant and animal cells. The tRNA cytokinins are synthesized by modification of spe-cific adenine residues within the fully transcribed tRNA. As with the free cytokinins, isopentenyl groups are trans-ferred to the adenine molecules from DMAPP by an enzyme call tRNA-IPT. The genes for tRNA-IPT have been cloned from many species.Cytokinins:Regulators of Cell Division499 ChromosomalCrown gallFIGURE 21.5 Agrobacterium tumefaciens a nitrogen source.The possibility that free cytokinins are derived from tRNA has been explored extensively. Although the tRNA-bound cytokinins can act as hormonal signals for plant cells if the tRNA is degraded and fed back to the cells, it is unlikely that any significant amount of the free hormonal cytokinin in plants is derived from the turnover of tRNA.An enzyme with IPT activity was identified from crude extracts of various plant tissues, but researchers were unable to purify the protein to homogeneity. Recently, plant IPT genes were cloned after the Arabidopsis genome was analyzed for potential ipt-like sequences (Kakimoto 2001; Takei et al. 2001). Nine different IPT genes were identifiedin Arabidopsis—many more than are present in animal genomes, which generally contain only one or two such genes used in tRNA modification.Phylogenetic analysis revealed that one of the Arabidop-sis IPT genes resembles bacterial tRNA-ipt, another resem-bles eukaryotic tRNA-IPT, and the other seven form a dis-tinct group or clade together with other plant sequences (see Web Topic 21.6). The grouping of the seven Arabidop-sis IPT genes in this unique plant clade provided a clue that these genes may encode the cytokinin biosynthetic enzyme.The proteins encoded by these genes were expressed in E. coli and analyzed. It was found that, with the exception of the gene most closely related to the animal tRNA-IPT genes, these genes encoded proteins capable of synthesiz-ing free cytokinins. Unlike their bacterial counterparts, how-ever, the Arabidopsis enzymes that have been analyzed uti-lize ATP and ADP preferentially over AMP(see Figure 21.6). Cytokinins from the Root Are Transportedto the Shoot via the XylemRoot apical meristems are major sites of synthesis of the free cytokinins in whole plants. The cytokinins synthesized in roots appear to move through the xylem into the shoot, along with the water and minerals taken up by the roots. This pathway of cytokinin movement has been inferred from the analysis of xylem exudate.When the shoot is cut from a rooted plant near the soil line, the xylem sap may continue to flow from the cut stump for some time. This xylem exudate contains cyto-kinins. If the soil covering the roots is kept moist, the flow of xylem exudate can continue for several days. Because the cytokinin content of the exudate does not diminish, the cytokinins found in it are likely to be synthesized by the roots. In addition, environmental factors that interfere with root function, such as water stress, reduce the cytokinin content of the xylem exudate (Itai and Vaadia 1971). Con-versely, resupply of nitrate to nitrogen-starved maize roots results in an elevation of the concentration of cytokinins in the xylem sap (Samuelson 1992), which has been correlated to an induction of cytokinin-regulated gene expression in the shoots (Takei et al. 2001).Although the presence of cytokinin in the xylem is well established, recent grafting experiments have cast doubt on the presumed role of this root-derived cytokinin in shoot development. Tobacco transformed with an inducible ipt gene from Agrobacterium displayed increased lateral bud outgrowth and delayed senescence.To assess the role of cytokinin derived from the root, the tobacco root stock engineered to overproduce cytokinin was grafted to a wild-type shoot. Surprisingly, no pheno-typic consequences were observed in the shoot, even though an increased concentration of cytokinin was mea-sured in the transpiration stream (Faiss et al. 1997). Thus the excess cytokinin in the roots had no effect on the grafted shoot.Roots are not the only parts of the plant capable of syn-thesizing cytokinins. For example, young maize embryos synthesize cytokinins, as do young developing leaves, young fruits, and possibly many other tissues. Clearly, fur-ther studies will be needed to resolve the roles of cytokinins transported from the root versus cytokinins synthesized in the shoot.A Signal from the Shoot Regulates the Transport of Zeatin Ribosides from the RootThe cytokinins in the xylem exudate are mainly in the form of zeatin ribosides. Once they reach the leaves, some of these nucleosides are converted to the free-base form or to glucosides (Noodén and Letham 1993). Cytokinin glu-cosides may accumulate to high levels in seeds and in leaves, and substantial amounts may be present even in senescing leaves. Although the glucosides are active as cytokinins in bioassays, often they lack hormonal activ-ity after they form within cells, possibly because they are compartmentalized in such a way that they are unavail-able. Compartmentation may explain the conflicting obser-vations that cytokinins are transported readily by the xylem but that radioactive cytokinins applied to leaves in intact plants do not appear to move from the site of appli-cation.Evidence from grafting experiments with mutants sug-gests that the transport of zeatin riboside from the root to the shoot is regulated by signals from the shoot. The rms4 mutant of pea (Pisum sativum L.) is characterized by a 40-fold decrease in the concentration of zeatin riboside in the xylem sap of the roots. However, grafting a wild-type shoot onto an rms4mutant root increased the zeatin riboside lev-els in the xylem exudate to wild-type levels. Conversely, grafting an rms4mutant shoot onto a wild-type root low-ered the concentration of zeatin riboside in the xylem exu-date to mutant levels (Beveridge et al. 1997).These results suggest that a signal from the shoot can regulate cytokinin transport from the root. The identity of this signal has not yet been determined.Cytokinins Are Rapidly Metabolized byPlant TissuesFree cytokinins are readily converted to their respective nucleoside and nucleotide forms. Such interconversions likely involve enzymes common to purine metabolism.Many plant tissues contain the enzyme cytokinin oxi-dase, which cleaves the side chain from zeatin (both cis and trans), zeatin riboside, iP, and their N-glucosides, but not their O-glucoside derivatives (Figure 21.7). However, dihydrozeatin and its conjugates are resistant to cleavage. Cytokinin oxidase irreversibly inactivates cytokinins, and it could be important in regulating or limiting cytokinin effects. The activity of the enzyme is induced by high cytokinin concentrations, due at least in part to an eleva-tion of the RNA levels for a subset of the genes.Cytokinins:Regulators of Cell Division501。