Unique and overlapping expression patterns among the Arabidopsis 1-amino-cyclopropane-1-carboxylate synthase gene family members

1-Aminocyclopropane-1-carboxylate synthase (ACS) catalyzes the rate-limiting step in the ethylene biosynthetic pathway in plants. The Arabidopsis genome encodes nine ACS polypeptides that form eight functional (ACS2, ACS4-9, and ACS11) homodimers and one nonfunctional (ACS1) homodimer. Transgenic Arabidopsis lines were constructed expressing the beta-glucuronidase (GUS) and green fluorescence protein (GFP) reporter genes from the promoter of each of the gene family members to determine their patterns of expression during plant development. All genes, except ACS9, are expressed in 5-d-old etiolated or light-grown seedlings yielding distinct patterns of GUS staining. ACS9 expression is detected later in development. Unique and overlapping expression patterns were detected for all the family members in various organs of adult plants. ACS11 is uniquely expressed in the trichomes of sepals and ACS1 in the replum. Overlapping expression was observed in hypocotyl, roots, various parts of the flower (sepals, pedicle, style, etc.) and in the stigmatic and abscission zones of the silique. Exogenous indole-3-acetic acid (IAA) enhances the constitutive expression of ACS2, 4, 5, 6, 7, 8, and 11 in the root. Wounding of hypocotyl tissue inhibits the constitutive expression of ACS1 and ACS5 and induces the expression of ACS2, 4, 6, 7, 8, and 11. Inducers of ethylene production such as cold, heat, anaerobiosis, and Li(+) ions enhance or suppress the expression of various members of the gene family in the root of light-grown seedlings. Examination of GUS expression in transverse sections of cotyledons reveals that all ACS genes, except ACS9, are expressed in the epidermis cell layer, guard cells, and vascular tissue. Similar analysis with root tip tissue treated with IAA reveals unique and overlapping expression patterns in the various cell types of the lateral root cap, cell division, and cell expansion zones. IAA inducibility is gene-specific and cell type-dependent across the root tip zone. This limited comparative exploration of ACS gene family expression reveals constitutive spatial and temporal expression patterns of all gene family members throughout the growth period examined. The unique and overlapping gene activity pattern detected reveals a combinatorial code of spatio-temporal coexpression among the various gene family members during plant development. This raises the prospect that functional ACS heterodimers may be formed in planta.     

混播草地不同种群再生性的研究

在不同刈割频率和时间尺度下 ,对混播草地多年生黑麦草 (Lolium perenne)分蘖数和叶片生长、白三叶 (Trifoliumrepens)分枝数和匍匐茎生长及不同种群年产量和组分进行了连续 3年的监测研究 .结果表明 ,刈割能刺激黑麦草叶片、白三叶匍匐茎生长和分枝数发生 ,保持混播草地黑麦草和白三叶的适宜比和稳定共存 ,提高草地年生产力 ,但不同刈割频率和刈割时间对其影响差异不显著 (P >0 .0 5 ) .黑麦草叶片生长对 6月刈割效果比 8月明显 ,而白三叶匍匐茎生长则与之相反 ,黑麦草产量主要取决于叶片生长 ,白三叶产量主要取决于匍匐茎分枝数 .刈割的黑麦草、白三叶产量组分比分别为 5 0 %、15 % ,比试验前约低 10 %、5 % ,而CK为 39%、6 % .     

Heritability of,and relationships between phosphorus and nitrogen concentration in shoot,stolon and root of white clover (Trifolium repens L.)

Ninety eight white clover genotypes were cloned and grown in pots at two levels of phosphorus (P) supply in soil. After harvest the nitrogen (N) and P content of shoot (leaf, petiole and unrooted stolon), stolon and root tissue was determined. Broad sense heritabilities for %N, %P, and proportion of total N or P in each tissue type were calculated. Heritabilities ranged from 0.22 to 0.68. They were generally higher for %P than %N; and higher in shoot and stolon tissue than root tissue for %P, %N, and proportion of N or P. Level of P in which plants were grown had little effect on heritability values. Genotypes from bred cultivars differed from those collected from hill country pastures for plant size, and partitioning of N and P to shoot, stolon and root. Relationships between plant characters were examined to determine the consequences of selection.     

Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8

Apically expressed intestinal and renal sodium-hydrogen exchangers (NHEs) play a major role in Na(+) absorption. Our previous studies on NHE ontogeny have shown that NHE-2 and NHE-3 are expressed at very low levels in young animals. Furthermore, single and/or double NHE-2 and NHE-3 knockout mice display no obvious abnormalities before weaning. These observations suggest that other transporter(s) may be involved in intestinal Na+ absorption during early life. The present studies were designed to clone the novel rat intestinal NHE-8 cDNA and to decipher the NHE-8 protein localization and gene expression pattern during different developmental stages. The rat NHE-8 cDNA has 2,160 bp and encodes a 575-amino acid protein. An antibody against NHE-8 protein was developed. Immunohistochemistry staining indicated apical localization of NHE-8 protein in rat intestinal epithelial cells. The apical localization of NHE-8 was also confirmed by its presence in brush-border membrane and its absence in basolateral membrane preparations. Northern blotting utilizing a NHE-8-specific probe demonstrated higher NHE-8 mRNA expression in young animals compared with adult animals. Western blot analysis revealed a similar pattern. Tissue distribution with multiple human tissue RNA blot showed that NHE-8 was expressed in multiple tissues including the gastrointestinal tract. In conclusion, we have cloned the full-length NHE-8 cDNA from rat intestine and further showed its apical localization in intestinal epithelial cells. We have also shown that NHE-8 gene expression and protein expression were regulated during ontogeny. Our data suggests that NHE-8 may play an important role in intestinal Na+ absorption during early life.     

Transformation of the forage legume Trifolium repens L. using binary Agrobacterium vectors

A system was established for introducing cloned genes into white clover (Trifolium repens L.). A high regeneration white clover genotype was transformed with binary Agrobacterium vectors containing a chimaeric gene which confers kanamycin resistance. Transformed kanamycin resistant callus was obtained by culturing Agrobacterium inoculated stolon internode segments on selective medium. The kanamycin resistance phenotype was stable in cells and in regenerated shoots. Transformation was confirmed by the expression of an unselected gene, nopaline synthase in selected cells and transgenic shoots and by the detection of neomycin phosphotransferase II enzymatic activity in kanamycin resistant cells. Integration of vector DNA sequences into plant DNA was demonstrated by Southern blot hybridisation.     

Differential expression of four genes encoding 1-aminocyclopropane-1-carboxylate synthase in Lupinus albus during germination, and in response to indole-3-acetic acid and wounding

1-Aminocyclopropane-1-carboxylate (ACC) synthase (ACS; EC 4.4.1.14) is the key regulatory enzyme of the ethylene biosynthetic pathway and is encoded by a multigene family in Arabidopsis thaliana, tomato, mung bean and other plants. Southern blot analysis revealed the existence of at least five ACS genes in white lupin (Lupinus albus L.) genome. Four complete and one partial sequences representing different ACS genes were cloned from the lupin genomic library. The levels of expression of two of the genes, LA-ACS1 and LA-ACS3, were found to increase after hypocotyl wounding. Apparently, these two genes were up-regulated by exogenous IAA treatment of seedlings. The LA-ACS3 mRNA levels were also elevated in the apical part of hypocotyl, which is reported to contain a high endogenous auxin concentration. This gene may be involved in the auxin- and ethylene-controlled apical hook formation. The expression of the LA-ACS4 gene was found to be almost undetectable. This gene may represent a “silent” twin of LA-ACS5 as these two genes share a considerable level of homology in coding and non-coding regions. The LA-ACS5 mRNA is strongly up-regulated in the embryonic axis of germinating seeds at the time of radicle emergence, and was also found in roots and hypocotyls of lupin seedlings. Received: 19 July 1999 / Accepted: 3 March 2000     

Adipokine gene expression in dog adipose tissues and dog white adipocytes differentiated in primary culture.

Obesity and its associated disorders are increasing in companion animals, particularly in dogs. We have investigated whether genes encoding key adipokines, some of which are implicated in the pathologies linked to obesity, are expressed in canine adipose tissues. Using RT-PCR, mRNAs encoding the following adipokines were detected in dog white adipose tissue: adiponectin, leptin, angiotensinogen, plasminogen activator inhibitor-1, IL-6, haptoglobin, metallothionein-1 and 2, and nerve growth factor. The adipokine mRNAs were present in all fat depots examined. Fractionation of adipose tissue by collagenase digestion showed that each gene was expressed in mature adipocytes. The mRNA for TNFalpha was not evident in adipose tissue, but was detected in isolated adipocytes. Fibroblastic preadipocytes from gonadal white fat were differentiated into adipocytes in primary culture and adipokine expression examined before and after differentiation (days 0 and 11, respectively). Each adipokine gene expressed in dog white adipocytes was also expressed in the differentiated cells. These results demonstrate that dog white adipose tissue expresses major adipokine genes, expression being in the adipocytes. Investigation of adipokine production and function will provide insight into the mechanisms involved in obesity-related pathologies in dogs and serve as a model for the related human diseases.     

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.     

The Influence of Leaf Position and Defoliation on the Assimilation and Translocation of Carbon in White Clover (Trifolium repens L.) 1. Carbon Distribution Patterns

The assimilation of carbon (C) by, and distribution of ¹⁴C from,leaves at each end of an unbroken sequence of ten mature leaveson the main stolon of clonal plants of white clover (Trifoliumrepens L.) were measured to identify intra-plant factors determiningthe direction of C movement from leaves. Leaves at two intermediatepositions were also measured. Localized movement of ¹⁴C to sinks at the same node as, or atthe one to two nodes immediately behind, the fed leaf accountedfor 40–50% of the total ¹⁴C exported by all measured leaves.A further 50–60% of exported ¹⁴C was therefore availablefor more-distant sinks, and the direction of translocation ofthis C was determined by the relative total strength or demand(number x size x rate of activity or growth) of sinks forwardof, or behind, the leaf in question. Thus 85% of the ¹⁴C exportedfrom the youngest measured leaf moved toward the base of thestolon, while about 60% of the ¹⁴C exported from the oldestleaf moved acropetally. Defoliating plants to leave just one mature leaf on the mainstolon (at any one of the same four positions studied in undefoliatedplants), and no leaves on branches, resulted in: (1) increasednet photosynthetic rate in all residual leaves: (2) increased%export of fixed C from one of the four leaves; (3) increasedexport to the main stolon apex from all except the eldest leaf;(4) increased export to branches from three of the four leaves;and (5) decreased export to stolon tissue and roots from allleaves, within 3 d of defoliation. These responses would seemto ensure the fastest possible replacement of lost leaf areaand, thus, restoration of homeostatic growth. The observed patternsof C assimilation and distribution in both undefoliated anddefoliated white clover plants are consistent with the generalrules of source-sink theory; the distance between sources andcompeting sinks, and relative sink strength, emerge as the mostimportant intra-plant factors governing C movement. These resultsemphasize the need to consider plant morphology, and the modularnature of plant growth, when interpreting patterns of resourceallocation in clonal plants, or plant responses to stressessuch as partial defoliation. Trifolium repens L, white clover, photosynthesis, assimilate translocation, defoliation     

Identification and immunocytochemical analysis of DCNP1, a dendritic cell-associated nuclear protein.

Dendritic cells (DCs) are potent antigen-presenting cells (APCs). Among so-called professional APCs, only DCs can activate naive T cells to initiate immune response. To better understand molecular mechanisms underlying unique functions of DCs, we searched for genes specifically expressed in human DCs, using PCR-based cDNA subtraction in conjunction with differential screening. cDNAs generated from CD34(+) stem cell-derived CD1a(+) DC were subtracted with cDNA from monocytes and used for generation of a cDNA library. The cDNA library was differentially screened to select genes expressed in DCs more abundantly than in monocytes. We identified a gene encoding a protein composed of 244 amino acids, which we designated as DCNP1 (dendritic cell nuclear protein 1). In Northern blot analysis, DCNP1 mRNA was highly expressed in mature DCs and at a lower level in immature DCs. In contrast, monocytes and B cells do not express the gene. In multiple human tissue Northern blot analysis, expression of DCNP1 was detected in brain and skeletal muscle. To examine subcellular localization of DCNP1, we performed immunofluorescence analysis using an anti-DCNP1 polyclonal antibody and found the molecule to be localized mainly in the perinucleus. In an immunohistochemical analysis, we compared the expression of DCNP1 with CD68, a marker for DCs and macrophages, in spleen, lymph node, liver, and brain. While DCNP1-positive cells showed a similar tissue distribution to CD68-positive cells, the number of DCNP1-positive cells was much smaller than that of CD68-positive cells. Our findings are consistent with the proposal that DCNP1 is specifically expressed in DCs.     

Gene-associated CpG islands and the expression pattern of genes in rice.

In an attempt to understand the role of gene-associated CpG islands in the expression of plant genes, I determined the position of CpG islands within their associated genes and the expression of the genes in rice tissues. I examined the expression patterns of 75 rice genes by Northern hybridization analysis using RNAs isolated from four rice tissues: leaf, root, callus, and panicle at flowering stage. From the results of this analysis, I classified most of the genes into one of two groups: expression in a single tissue and expression in two or more tissues. There was a marked correlation between the expression of a gene in two or more tissues and the presence of a CpG island in its 5'-end (class 1 CpG island). Among the genes expressed in a single tissue, the genes expressed in callus were distinct from those expressed in other tissues in that a large proportion contained a class 1 CpG island. These results suggest that plant CpG islands may be useful for deducing the expression pattern of uncharacterized genes.     

A potato cDNA encoding a homologue of mammalian multidrug resistant P-glycoprotein

A homologue of the multidrug resistance (MDR) gene was obtained while screening a potato stolon tip cDNA expression library with³⁵S-labeled calmodulin. The mammalian MDR gene codes for a membrane-bound P-glycoprotein (170–180 kDa) which imparts multidrug resistance to cancerous cells. The potato cDNA (PMDR1) codes for a polypeptide of 1313 amino acid residues (ca. 144 kDa) and its structural features are very similar to the MDR P-glycoprotein. The N-terminal half of the PMDR1-encoded protein shares striking homology with its C-terminal half, and each half contains a conserved ATP-binding site and six putative transmembrane domains. Southern blot analysis indicated that potato has one or two MDR-like genes. PMDR1 mRNA is constitutively expressed in all organs studied with higher expression in the stem and stolon tip. The PMDR1 expression was highest during tuber initiation and decreased during tuber development.