Over-expression of tobacco knotted1-type class1 homeobox genes alters various leaf morphology

We compared the phenotypes of transgenic tobacco plants over-expressing various knotted1-type class1 homeobox genes. All transformants showed abnormal leaf morphology, with the degree of abnormality depending upon the Nicotiana tabacum homeobox (NTH) gene that was over-expressed. Tobacco plants over-expressing NTH1 or NTH9 showed a relatively weak phenotype, while NTH15 and NTH20 over-expressing plants exhibited severe alterations, with occasional ectopic shoot formation on the leaves. Plants over-expressing NTH22 had a relatively severe phenotype, but did not form any ectopic shoots. These results indicate that all of the NTH genes can influence leaf development from the shoot apical meristem, but that the effect varies with the gene. Based on phylogenetic analysis of the NTH genes and comparison of the phenotypes of plants over-expressing them, we suggest that the kn1-type class1 family can be divided into two subgroups, and that the differences in their ability to induce the abnormal phenotype corresponds to the structures of their conserved domains.     

Expression of class I knotted1-like homeobox genes in the storage roots of sweetpotato (Ipomoea batatas)

As a first step in clarifying the involvement of class I knotted1-like homeobox (KNOXI) genes in the storage root development of sweetpotato (Ipomoea batatas), we isolated three KNOXI genes, named Ibkn1, Ibkn2 and Ibkn3, expressed in the storage roots. Phylogenetic analysis showed that Ibkn1 was homologous to the SHOOT MERISTEMLESS (STM) gene of Arabidopsis, while Ibkn2 and Ibkn3 were homologous to the BREVIPEDICELLUS (BP) gene. Of these, expression of Ibkn1 and Ibkn2 were upregulated in developing and mature storage roots compared with fibrous roots. Ibkn1 and Ibkn2 showed different expression patterns in the storage roots. Ibkn1 was preferentially expressed at the proximal end and around the primary vascular cambium, while Ibkn2 expression was highest in the thickest part and lower in both the proximal and distal ends. In contrast to Ibkn1 and Ibkn2, expression of Ibkn3 in roots was not consistent among sweetpotato cultivars. The distribution of endogenous trans-zeatin riboside (t-ZR) in sweetpotato roots showed a similarity to the expression pattern of KNOXI genes, supporting the idea that KNOXI genes control cytokinin levels in the storage roots. The physiological functions of these KNOXI genes in storage root development are discussed.     

Ectopic expression of maize knotted1 results in the cytokinin-autotrophic growth of cultured tobacco tissues

The ectopic expression of knotted homologues has cytokinin-like effects on plant morphology. The functional relationship between knotted and cytokinins was investigated in cultures of leaf tissue established from tobacco (Nicotiana tabacum L. cv. Havana 425) plants transformed with the maize knotted1 (kn1) gene regulated by cauliflower mosaic virus 35S RNA expression signals. In contrast to leaf tissues of untransformed plants, leaf tissues of kn1 transformants were capable of sustained, cytokinin-autotrophic growth on auxin-containing medium and resembled the tobacco cytokinin-autotrophic mutants Hl-1 and Hl-2. The concentration of 18 cytokinins was measured in cultures initiated from leaves of three independent kn1 transformants and the Hl-1 and Hl-2 mutants. Although cytokinin contents were variable, the content of several cytokinins in Kn1, Hl-1 and Hl-2 tissue lines was at least 10-fold higher than that of wild-type tobacco tissues and in the range reported for other cytokinin-autotrophic tobacco tissues. These results suggest that the cytokinin-autotrophic growth of Kn1 lines could result from elevated steady-state levels of cytokinins. Received: 7 July 1999 / Accepted: 10 November 1999     

Leaf senescence is delayed in tobacco plants expressing the maize homeobox gene knotted1 under the control of a senescence-activated promoter.

Leaf senescence is an active process involving remobilization of nutrients from senescing leaves to other parts of the plant. Whereas senescence is accompanied by a decline in leaf cytokinin content, supplemental cytokinin delays senescence. Plants that overexpress isopentenyl transferase (ipt), a cytokinin-producing gene, or knotted1 (kn1), a homeobox gene, have many phenotypes in common. Many of these phenotypes are characteristic of altered cytokinin physiology. The effect of kn1 on leaf senescence was tested by driving its expression using the promoter of the senescence-associated gene SAG12. SAG:kn1 tobacco plants showed a marked delay in leaf senescence but otherwise developed normally. The delay in senescence was revealed by an increase in chlorophyll content in SAG:kn1 leaves relative to leaves of the control plants and by a decrease in the number of dead leaves. Senescence was also delayed in detached leaves of SAG:kn1 plants. Delayed senescence was accompanied by increased leaf cytokinin content in older leaves expressing kn1. These experiments extend the current understanding of kn1 function and suggest that in addition to mediating meristem maintenance, kn1 is capable of regulating the onset of senescence in leaves.     

Regulation of CLV3 expression by two homeobox genes in Arabidopsis

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. The WUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3 (CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes in CLV3 expression levels, and adjustment of WUS expression via the CLV signal transduction pathway. We have analyzed whether expression of CLV3 is controlled by the activity of WUS or another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends on WUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level of CLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Regional expression of the rice KN1-type homeobox gene family during embryo, shoot, and flower development.

We report the isolation, sequence, and pattern of gene expression of members of the KNOTTED1 (KN1)-type class 1 homeobox gene family from rice. Phylogenetic analysis and mapping of the rice genome revealed that all of the rice homeobox genes that we have isolated have one or two direct homologs in maize. Of the homeobox genes that we tested, all exhibited expression in a restricted region of the embryo that defines the position at which the shoot apical meristem (SAM) would eventually develop, prior to visible organ formation. Several distinct spatial and temporal expression patterns were observed for the different genes in this region. After shoot formation, the expression patterns of these homeobox genes were variable in the region of the SAM. These results suggest that the rice KN1-type class 1 homeobox genes function cooperatively to establish the SAM before shoot formation and that after shoot formation, their functions differ.     

The expression of LIM‐homeobox genes,Lhx1 and Lhx5, in the forebrain is essential for neural retina differentiation

Elucidating the mechanisms underlying eye development is essential for advancing the medical treatment of eye‐related disorders. The primordium of the eye is an optic vesicle (OV), which has a dual potential for generation of the developing neural retina and retinal pigment epithelium. However, the factors that regulate the differentiation of the retinal primordium remain unclear. We have previously shown that overexpression of Lhx1 and Lhx5, members of the LIM‐homeobox genes, induced the formation of a second neural retina from the presumptive pigmented retina of the OV. However, the precise timing of Lhx1 expression required for neural retina differentiation has not been clarified. Moreover, RNA interference of Lhx5 has not been previously reported. Here, using a modified electroporation method, we show that, Lhx1 expression in the forebrain around stage 8 is required for neural retina formation. In addition, we have succeeded in the knockdown of Lhx5 expression, resulting in conversion of the neural retina region to a pigment vesicle‐like tissue, which indicates that Lhx5 is also required for neural retina differentiation, which correlates temporally with the activity of Lhx1. These results suggest that Lhx1 and Lhx5 in the forebrain regulate neural retina differentiation by suppressing the development of the retinal pigment epithelium, before the formation of the OV.     

Distinct structural domains in the planarian brain defined by the expression of evolutionarily conserved homeobox genes

 Homeobox genes such as orthodenticle in Drosophila and its mouse homologues, Otx1 and Otx2, are known to be essential for rostral brain development. To investigate the molecular basis of brain evolution, we searched for otd/Otx-related homeobox genes in the planarian Dugesia japonica, and identified two genes, DjotxA and B, whose expression appears to be restricted to the cephalic ganglion (brain). DjotxA was expressed more medially, in the region containing the termini of the visual axons, and in the visual cells, suggesting involvement in establishment of the visual system. DjotxB was expressed in a discrete region just lateral to the DjotxA-positive domain, but not in the more lateral branch structures, which in turn are characterized by the expression of Djotp, a planarian homeobox gene related to mouse Orthopedia (Otp). In transverse sections of planarians, DjotxA and B expression were observed only at the anterior ends of the stumps, corresponding to the regional pattern of the regenerating brain. Our findings suggest that the planarian brain is composed of structurally distinct and functionally diverse domains which are defined by the discrete expression of the three evolutionarily conserved homeobox genes. Received: 17 June 1998 / Accepted: 20 August 1998     

Abscisic acid regulates TSRF1-mediated resistance to Ralstonia solanacearum by modifying the expression of GCC box-containing genes in tobacco

Although recent studies have established a significant regulatoryrole for abscisic acid (ABA) and ethylene response factor (ERF)proteins in plant pathogen resistance, it is not clear whetherand how ABA performs this role. Previously, it was reportedthat an ERF protein, TSRF1, activates the expression of GCCbox-containing genes and significantly enhances the resistanceto Ralstonia solanacearum in both tobacco and tomato plants.Here, it is reported that TSRF1-regulated pathogen resistanceis modified by ABA application. TSRF1 activates the expressionof ABA biosynthesis-related genes, resulting in the increaseof ABA biosynthesis, which further stimulates ethylene production.More interestingly, ABA application decreases, while the inhibitorof ABA biosynthesis fluridone increases, the TSRF1-enhancedresistance to R. solanacearum. This observation is further supportedby the finding that ABA and fluridone reversibly modify theability of TSRF1 to bind the ethylene-responsive GCC box, consequentlyaltering the expression of element-controlled genes. These resultstherefore establish that TSRF1-regulated resistance to R. solanacearumcan be modified in tobacco by ABA. Key words: Abscisic acid, ERF protein TSRF1, GCC box-containing genes, Ralstonia solanacearum, tobacco     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.     

Protein dephosphorylation mediates salicylic acid-induced expression of PR-1 genes in tobacco

Several lines of evidence suggest that salicylic acid (SA) is an endogenous signal for the activation of several plant defense responses, including the expression of genes encoding pathogenesis-related (PR) proteins such as the acidic PR-1 proteins. During recent years, studies have suggested that interaction of SA with catalase and ascorbate peroxidase leads to two signals in tobacco - elevated H₂O₂ levels and lipid peroxides. However, to date, relatively little is known about the molecular and biochemical mechanisms that mediate transduction beyond these signals or through other SA-effector proteins. Using protein kinase and phosphatase inhibitors, this study demonstrates that PR-1 gene induction can be mediated by dephosphorylation of serine/threonine residue(s) of two or more unidentified phosphoproteins. The protein phosphatase inhibitors, okadaic acid and calyculin A blocked SA-mediated induction of PR-1 genes, implying the involvement of a phosphoprotein downstream of SA. The protein kinase inhibitors K-252a and staurosporine induced PR-1 gene expression. PR-1 gene induction by K-252a was suppressed by okadaic acid. Surprisingly, this induction was also suppressed in NahG transgenic tobacco plants which convert SA to catechol. Moreover, K-252a stimulated production of SA and its glucoside, suggesting that another phosphoprotein acts upstream of SA. Taken together, these results suggest that there are two (or more) phosphoproteins which function in the same signal transduction pathway leading to PR-1 gene induction. The SA-inducible acidic PR-2 genes were similarly affected by the inhibitors, while the genes for actin and phenylalanine ammonia lyase were not.     

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox geneOSH1 lead to altered morphology of leaves in transgenic tobacco

Transgenic tobacco plants were generated carrying a rice homeobox gene,OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicated arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotropic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly,OSH1 expression was undetectable in mature leaves with the aberrant morphological features. Thus,OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate thatPR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopicOSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. TheOSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.     

Distal regulatory regions restrict the expression of cis-linked genes to the tapetal cells

Rapid light-dependent turnover of the chloroplast-encoded D1 protein maintains photosystem II (PS II) functional over a wide range of light intensities. Following initiation of psbA mRNA translation, the elongating D1 is targeted, possibly by chloroplast signal recognition particle 54 (cpSRP54), to the thylakoid cpSecY translocation channel. Transmembrane domains of nascent D1 start interacting with other PS II core proteins already during the translocation process to ensure an efficient assembly of the multiprotein membrane complex. Here we review the progress recently made concerning the synthesis, targeting, membrane insertion and assembly to PS II of the chloroplast-encoded D1 protein and discuss the possible convergence of targeting and translocation of chloroplast- and nuclear-encoded thylakoid proteins.