Cambial Region Research Articles

The ectopic expression of PttKN1 gene causes pleiotropic alternation of morphology in transgenic carnation (Dianthus caryophyllus L.)

Carnation (Dianthus caryophyllus L.) is one of the most important ornamental plants in the world. Though morphological modification of carnation is very important to its commercial value, there have been no relevant reports until now. PttKN1 (Populus tremula × Tremuoides knotted1), isolated from the vascular cambial region of hybrid aspen, is a novel member of KNOX gene family. In this paper, we transformed 35S:PttKN1 to carnation via Agrobacterium tumefaciens. All primary transformants subsequently obtained were placed into phenotypic categories and self-pollinated. A total of 32 T0 progeny with aberrant phenotypes were obtained. PCR assay proved the validity of these transgenic plants. Phenotypes of 32 35S:PttKN1 plants were distinct from those of wild-type plants, including: (1) modification of phyllotaxis (15/32): wild-type carnation was with typical opposite phyllotaxis, while transgenic plants displayed tricussate whorled and multiple-cussate whorled phyllotaxis. Irregular modification of phyllotaxis was also observed; (2) modification of stem (9/32): wild-type stems were round; however, some transgenic plants exhibited much thicker and flatter stem; (3) the whole transgenic plants of carnation (8/32) became dwarf. These morphological modifications of carnation indicate that we have successfully attained some novel lines of carnation. These lines can have potential practical applications. In conclusion, the selection of stably genetic lines is discussed.

In vitro organogenesis of Citrus volkameriana and Citrus aurantium

In vitro organogenesis of Citrus volkameriana and C. aurantium was studied considering three explant types: epicotyl segment, internodal segment, and hypocotyl segment with attached cotyledon fragment. The explants were cultured in medium according to Grosser and Gmitter (EME) supplemented with 0, 0.5, 1.0, 1.5, and 2.0 mg dm− 3 6-benzyl-aminopurine (BAP), incubated firstly in darkness for 4 weeks, and then transferred to 16-h photoperiod for 2 weeks. Comparing epicotyl and internodal segments, a higher percentage of responsive explants and a higher number of shoots per explant were obtained with epicotyl segments, regardless of the BAP concentration. For C. volkameriana the highest percentage of responsive epicotyl segments (42 %) was obtained in EME with 1.0 mg dm−3 BAP, while for C. aurantium (59 %) in EME with 0.5 mg dm−3 BAP. The organogenesis efficiency was the best with the use of the hypocotyl segment with attached cotyledon fragment (77 % for C. volkameriana and to 75 % for C. aurantium). With this explant the morphogenesis occurred only in the hypocotyl region. The in vitro organogenesis was characterized by histological analyses showing that the morphogenic process started in the cambium region near the explant cut end.

Induction of PtoCDKB and PtoCYCB transcription by temperature during cambium reactivation in Populus tomentosa Carr.

Cell cycle progression requires interaction between cyclin-dependent kinase B (CDKB) and cyclin B (CYCB). The seasonal expression patterns of the CDKB and CYCB homologues from Populus tomentosa Carr. were investigated, and effects of temperature and exogenous indole-3-acetic acid (IAA) on their expression were further studied in water culture experiments. Based on the differential responses of dormant cambium cells to exogenous IAA, four stages of cambium dormancy were confirmed for P. tomentosa: quiescence 1 (Q1), rest, quiescence 2-1 (Q2-1), and quiescence 2-2 (Q2-2). PtoCDKB and PtoCYCB transcripts were strongly expressed in the active phases, weakly in Q1, and almost undetectable from rest until late Q2-2. Climatic data analysis showed a correlation between daily air temperature and PtoCDKB and PtoCYCB expression patterns. Water culture experiments with temperature treatment further showed that a low temperature (4 °C) kept PtoCDKB and PtoCYCB transcripts at undetectable levels, while a warm temperature (25 °C) induced their expression in the cambium region. Meanwhile, water culture experiments with exogenous IAA treatment showed that induction of PtoCDKB and PtoCYCB transcription was independent of exogenous IAA. The results suggest that, in deciduous hardwood P. tomentosa growing in a temperate zone, the temperature in early spring is a vital environmental factor for cambium reactivation. The increasing temperature in early spring may induce CDKB and CYCB homologue transcription in the cambium region, which is necessary for cambium cell division.

Transformation of PttKN1 gene to cockscomb

We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5 mg l−1 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l−1 6-BA, 0.5 mg l−1 NAA and 0.5 mg l−1 IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Proteomic analysis of the cambial region in juvenile Eucalyptus grandis at three ages

Recent advances in genomics and proteomics have provided an excellent opportunity to understand complex biological processes such as wood formation at the gene and protein levels. The aim of this work was to describe the proteins participating in the processes involved in juvenile wood formation by isolating proteins from the cambial region of Eucalyptus grandis, at three ages of growth (6-month-old seedlings, 3- and 6-year-old trees), and also to identify proteins differentially expressed. Using a 2-D-LC-MS/MS strategy we identified a total of 240 proteins, with 54 corresponding spots being present in at least two ages. Overall, nine proteins classified into the functional categories of metabolism, cellular processes, and macromolecular metabolism showed significant changes in expression. Proteins were classified into seven main functional categories, with metabolism representing 35.2% of the total proteins identified. The comparison of the reference maps showed not only differences in the expression pattern of individual proteins at each age, but also among isoforms. The results described in this paper provide a dynamic view of the proteins involved in the formation of juvenile wood in E. grandis.

ANAC012, a member of the plant‐specific NAC transcription factor family, negatively regulates xylary fiber development in Arabidopsis thaliana

Vascular plants evolved to have xylem that provides physical support for their growing body and serves as a conduit for water and nutrient transport. In a previous study, we used comparative-transcriptome analyses to select a group of genes that were upregulated in xylem of Arabidopsis plants undergoing secondary growth. Subsequent analyses identified a plant-specific NAC-domain transcription factor gene (ANAC012) as a candidate for genetic regulation of xylem formation. Promoter-GUS analyses showed that ANAC012 expression was preferentially localized in the (pro)cambium region of inflorescence stem and root. Using yeast transactivation analyses, we confirmed the function of ANAC012 as a transcriptional activator, and identified an activation domain in the C terminus. Ectopic overexpression of ANAC012 in Arabidopsis (35S::ANAC012 plants) dramatically suppressed secondary wall deposition in the xylary fiber and slightly increased cell-wall thickness in the xylem vessels. Cellulose compositions of the cell wall were decreased in the inflorescent stems and roots of 35S::ANAC012 plants, probably resulting from defects in xylary fiber formation. Our data suggest that ANAC012 may act as a negative regulator of secondary wall thickening in xylary fibers.

Coordination of PsAS1 and PsASPG expression controls timing of re-allocated N utilization in hypocotyls of pine seedlings

During pine seed germination, a large amount of N mobilized from the storage proteins is re-allocated in the hypocotyl as free asparagine, as a result of the high levels of asparagine synthetase (AS) encoded by the PsAS1 gene. To determine the role of this re-allocated N reserve, a full-length cDNA encoding L: -asparaginase (ASPG) has been cloned from Scots pine (Pinus sylvestris L.) seedlings and characterized. Like other N-terminal nucleophile hydrolases, pine ASPG requires a post-translational processing to exhibit enzymatic activity. However, in contrast to previous reports on other plant ASPGs, purified recombinant pine ASPG does not undergo autoproteolytic cleavage in vitro. Our results suggest that the processing requires accessory proteins to assist in the proteolysis or in the proper folding before autocleavage in a divalent cation-dependent manner. Sequence comparison analysis revealed that the pine protein is included in the K+-dependent subfamily of plant ASPGs. The expression of the ASPG-encoding gene (PsASPG) was higher in organs with extensive secondary development of the vascular system. The increase in transcript abundance observed at advanced stages of hypocotyl development was concomitant with a decrease of PsAS1 transcript abundance and a remarkable increase in the number of xylem elements and highly lignified cell walls. These results, together with the precise localization of PsASPG transcripts in cells of the cambial region, suggest that the expression of PsAS1 and PsASPG is temporally coordinated, to control the re-allocation of N from seed storage proteins toward the hypocotyl to be later used during early development of secondary vascular system.

Lignin composition in cambial tissues of poplar

The cambial tissues of a Populus balsamifera, Balsam poplar clone were studied during a growth season. The Klason and acid-soluble lignin contents were determined as well as the carbohydrate monomer distribution and the protein content. Both the phloem and the xylem sides of the cambial region were examined. The samples were analyzed by thioacidolysis and structures of dimeric products were determined by mass spectrometry after desulphuration. Chemical analysis of samples during the growth season was combined with microscopy of embedded specimens that showed the state of cell differentiation at the time of sampling. In spring and early summer, growth is very rapid and the intention was to collect tissue in which exclusively the middle lamella/primary cell wall had begun to lignify. The Klason lignin, protein content and carbohydrate monomer distribution showed that all the specimens from the cambial tissues sampled during a growth season contained predominantly middle lamella and primary walls; except for the developing xylem sampled in August where the carbohydrate composition showed that secondary walls were present. Thioacidolysis showed that the lignin from the cambial tissues had more condensed structures than the lignin from the reference balsam poplar clone wood. More guaiacyl than syringyl units were detected and mass spectrometry showed that the cambial tissues contained more lignin structures with end-groups than the reference sample. These results suggest that lignification in the cambial layer and early developing xylem may take place predominantly in a bulk fashion during the summer.

Regular cambial activity and xylem and phloem formation in locally heated and cooled stem portions of Norway spruce

The effect of heating (23–25°C) and cooling (9–11°C) on regular cambial activity and xylem and phloem formation in the stem portion of Norway spruce was investigated. Adult trees were sampled at 21-day intervals during the 2005 vegetation period. Continuously elevated temperatures increased the rate of cell division in the first part of the growing season, but did not significantly prolong cambial activity at the end of the vegetation period in the heated tree. Low-temperature treatments shortened regular cambial activity and slowed down cell production. The xylem growth ring was wider in the heated sample and narrower in the cooled sample compared to the control. The temperature in the cambial region was only negligibly transferred along the stem from the site of its application. In general, the temperature in the cambium was affected by a long-term rise or drop in air temperatures. Both experiments affected the structure and width of phloem growth increments. The tangential band of the axial parenchyma was not continuous in the cooled sample. The number of late phloem cells was reduced in the cool-treated sample and increased in the heat-treated sample. Our experiments confirmed the effect of constantly increased or decreased temperatures on regular cambial activity in Norway spruce.

ABP1 expression regulated by IAA and ABA is associated with the cambium periodicity in Eucommia ulmoides Oliv.

A cDNA clone of Eucommia ulmoides Oliv. encoding auxin binding protein 1 (ABP1), one of the putative receptors of auxin, was isolated, and the seasonal expression of ABP1 in relation to IAA and ABA annual variation was investigated by different technical approaches including RT-PCR, real-time PCR, northern blotting, western blotting, and immunolocalization. In the cambial region, ABP1 expression at both the protein and the mRNA level was found to be high, low, and remarkably scarce in the active, quiescent, and resting stages, respectively, during cambium periodicity. The signal abundance of ABP1 follows the opposite pattern to ABA accumulation and correlates with auxin responsiveness of the cambial tissues, suggesting a role for ABP1 in mediating auxin-dependent regulation of cambial activation in the activity-dormancy cycle. This paper attempts to explain why IAA would 'boost' the reactivation of a quiescent cambium, and not that of a resting cambium. Results also show that ABP1 expression is improved by IAA, while inhibited by ABA.

Histological characterization of in vitro adventitious organogenesis in Citrus sinensis

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm-3 benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.

Agrobacterium-mediated transformation of dormant lateral buds in poplar trees reveals developmental patterns in secondary stem tissues.

In an attempt to devise a method for the rapid creation of somatic transgenic wood sectors of sufficient size that would allow us to detect and analyse altered wood characteristics within them, we have explored the manual wounding and subsequent infection with Agrobacterium of dormant lateral buds in poplar. Following treatment and transformation with a 35S-GUS construct, frequent stable transformation was found in the form of distinct and specific GUS staining patterns in the outer cortex, cambial region (including primary and secondary xylem and phloem) and pith. Sector frequency and size were consistent with anatomical features of dormant lateral buds at the time of manual wounding and Agrobacterium-infection. The suitability of somatic sector analysis for functional genomic studies as well as for studies investigating pattern formation and the developmental fate of various cell-types within poplar stems is discussed.

Tissue‐specific localization of gibberellins and expression of gibberellin‐biosynthetic and signaling genes in wood‐forming tissues in aspen

Bioactive gibberellins (GAs) are known regulators of shoot growth and development in plants. In an attempt to identify where GAs are formed, we have analyzed the expression patterns of six GA biosynthesis genes and two genes with predicted roles in GA signaling and responses in relation to measured levels of GAs. The analysis was based on tangential sections, giving tissue-specific resolution across the cambial region of aspen trees (Populus tremula). Gibberellin quantification by GC/MS-SRM showed that the bioactive GA1 and GA4 were predominantly located in the zone of expansion of xylem cells. Based on co-localization of the expression of the late GA biosynthesis gene GA 20-oxidase 1 and bioactive GAs, we suggest that de novo GA biosynthesis occurs in the expanding xylem. However, expression levels of the first committed GA biosynthesis enzyme, ent-copalyl diphosphate synthase, were high in the phloem, suggesting that a GA precursor(s) may be transported to the xylem. The expression of the GA signaling and response genes DELLA-like1 and GIP-like1 coincided well with sites of high bioactive GA levels. We therefore suggest that the main role of GA during wood formation is to regulate early stages of xylem differentiation, including cell elongation.

Stem anatomy of Dolichos lablab Linn (Fabaceae): Origin of cambium and reverse orientation of vascular bundles

Secondary growth in the stem of Dolichos lablab is achieved by the formation of eccentric successive rings of vascular bundles. The stem is composed of parenchymatous ground tissue and xylem and phloem confined to portions of small cambial segments. However, development of new cambial segments can be observed from the obliterating ray parenchyma, the outermost phloem parenchyma and the secondary cortical parenchyma. Initially cambium develops as small segments, which latter become joined to form a complete cylinder of vascular cambium. Each cambial ring is functionally divided into two distinct regions. The one segment of cambium produces thick-walled lignified xylem derivatives in centripetal direction and phloem elements centrifugally. The other segment produces only thin-walled parenchyma on both xylem and phloem side. In mature stems, some of the axial parenchyma embedded deep inside the xylem acquires meristematic activity and leads to the formation of thick-walled xylem derivatives centrifugally and phloem elements centripetally. The secondary xylem comprises vessel elements, tracheids, fibres and axial parenchyma. Rays are uni-multiseriate in the region of cambium that produces xylem and phloem derivatives, while in some of the regions of cambium large multiseriate, compound, aggregate and polycentric rays can be noticed.

Ectopic Expression of the Pttknl Gene Induces Alterations in the Morphology of the Leaves and Flowers in Petunia hybrida Vilm.

Abstract: A novel knottedl‐like homeobox (knox) gene, Pttknl (Populus tremula tremuloides knottedl), isolated from the cambial region of hybrid aspen, was introduced into Petunia hybrida Vilm. using the leaf‐disc method mediated by Agrobacterium. A series of novel phenotypes was observed in transgenic petunia plants, including the formation of ectopic spikes on the adaxial surface of corollas and small pet als on the abaxial surface of corollas, fusion of floral organs, shortening of corolla midribs, the formation of tumor‐like knots along the midrib on the abaxial surface and serrated lobs of corolla margins, and alterations in pet al color; except for changes in the leaves and plant architecture, RT‐PCR showed that the Pttknl gene was expressed in the leaves of different petunia transgenic plants, whereas no signal was detected in wild‐type plants. The possible function of Pttknl in leaf and flower development is discussed.(Managing editor: Li‐Hui ZHAO)

Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division.

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

Extracellular sheath formation by Sphaeropsis hypodermia and association with its infection in elm trees

Sphaeropsis hypodermia, isolated from a cankered American elm branch, was grown on agar medium (PDA), on autoclaved wiping paper (Kimwipes), and American elm (Ulmus americana) wood chips, or inoculated into greenhouse-grown American elm saplings. Samples from each treatment were double-fixed with glutaraldehyde and osmium tetroxide and examined with the light and the transmission electron microscopes. Ultrastructurally, the hyphae on PDA and inert substrates appeared surrounded by large extracellular sheaths which were delimited by rigid opaque bands of various thicknesses. The sheaths extended appreciable distances from the fungal cells, as evidenced by their adherence to rigid substrates. Individual or aggregated opaque bodies, even as large masses on elm wood chips, were the main components of the sheath. This opaque material was often associated with penetration and ruptures of the wood cells. Inoculated into elm trees, the fungus rapidly caused pronounced alterations of cambial tissues and colonized the adjoining bark and xylem cells. The prominent penetration and breakdown of the inner and outer bark cells by the fungus were associated with opaque material, particularly in cortical fibres. The material was structurally similar to the sheath formed on the rigid sterilized substrates. In the xylem, only the walls of the recently deposited cells were visibly altered, and although mature fibres were generally colonized, the passage of the fungus from one fibre to another was rarely observed, contrary to the passage from vessel and ray cells to adjoining cells. In that instance, only bands of opaque material present in the walls of fibres were connected with fungal cells in their lumen. In the inner bark and cambial regions, cell hypertrophy and hyperplasia occurred next to host walls that were altered and contained similar opaque material. The extracellular sheath of S. hypodermia under in vitro conditions and the opaque material associated with host wall alterations in vivo are considered to be analogous.

Opposite patterns in the annual distribution and time-course of endogenous abscisic acid and indole-3-acetic acid in relation to the periodicity of cambial activity in Eucommia ulmoides Oliv.

The seasonal change of free abscisic acid (ABA) and indole-3-acetic acid (IAA) and their relationship with the cambial activity in Eucommia ulmoides trees were investigated by ABA and IAA immunolocalization using primary polyclonal and rhodamine-red fluorescing secondary antibodies, ABA and IAA quantification using high performance liquid chromatography (HPLC), and systematic monitoring of vascular cell layers production. ABA and IAA clearly displayed opposite annual distribution patterns. In the active period (AP), both immunolocalization and HPLC detected an abrupt decrease of ABA, reaching its lowest level in the summer. During dormancy, ABA started increasing in the first quiescence (Q1) (autumn), peaked in the rest (winter), and gradually decreased from the onset of the second quiescence (Q2) (the end of winter). IAA showed a reverse pattern to that of ABA: it sharply increased in AP, but noticeably decreased from the commencement of Q1. Longitudinally, the ABA distribution increased apico-basally, contrasting with IAA. Laterally, most of the ABA was located in mature vascular tissues, whereas the IAA essentially occurred in the cambial region. The concomitant IAA-ABA distribution and seasonal changes in vascular tissues greatly correlated with xylem and phloem cell production, and late wood differentiation and maturation. Interestingly, the application of exogenous ABA to quiescent E. ulmoides branches, in a water-culture system, inhibited external IAA action on cambial activity reactivation. These results suggest that, in E. ulmoides, ABA and IAA might probably interact in the cambial region. The annual cambial activity could be influenced by an IAA:ABA ratio; and ABA might play a key role in vascular cambium dormancy in higher plants. The relationship between hormonal changes and the (particular) annual life cycle of E. ulmoides is also discussed.