C. H. Anthony Little

Spatial pattern of cauliflower mosaic virus 35S promoter-luciferase expression in transgenic hybrid aspen trees monitored by enzymatic assay and non-destructive imaging

A protocol has been developed for efficiently transforming and regenerating the hybrid aspenPopulus tremula x P. tremuloides. Stem segments were co-cultivated with a strain ofAgrobacterium tumefaciens carrying a disarmed binary vector conferring resistance to kanamycin or hygromycin. The respective vectors also carried a fused bacterialluxF2 gene expressed from the cauliflower mosaic virus 35S promoter. All transformants had a normal phenotype. Genetic tranformation and stable integration of the heterologous DNA was confirmed by Southern hybridization and luciferase expression. The latter was measured by destructive enzymatic assay throughout the transformatnt and by non-destructive image analysis in leaves left attached to intact plants. Both measurement techniques detected marked within- and between-organ variation in luciferase expression. However, the spatial patterns detected by each technique in the leaves were similar. The results indicate thatin vivo imaging of light emission can be used to measure repeatedly the expression of a promoter-luciferase gene fusion in a particular leaf over an extended time period. It was also demonstrated that enzymatically assayed luciferase activity in leaves was notably lowere in transgenic hybrid aspen plants than in tobacco plants transformed with the same vector. This was not due to a difference in luciferase enzyme activity between the two species, and therefore indicated that the 35S promoter is not as active in hybrid aspen as in tobacco.

Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy.

We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen. Each promoter was fused to the uidA β-glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation. Both wildtype and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections. In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells. However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith. After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures. In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle. The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay. Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.

Enhanced ethylene production and peroxidase activity in IAA-overproducing transgenic tobacco plants is associated with increased lignin content and altered lignin composition

Abstract In a previous investigation, the lignin content of the xylem in the tobacco stem was shown to be greater in transgenic IAA-overproducing line C plants than in wild-type plants (Sitbon et al., Plant Physiol. 99 (1992) 1062–1069). Here, we confirm this observation and also show that the lignin composition in the transformants is altered, the ratio of syringyl to guaiacyl units being decreased due to an increase in guaiacyl units. Line C plants displayed an increased ethylene production in leaves and internodes, as well as a greater capacity to evolve ethylene in response to wounding and exogenous IAA. Line C plants also had greater peroxidase (POD) activity, whereas cinnamyl alcohol dehydrogenase and s-glucosidase activities were similar in the two genotypes. The mRNA level of a tobacco anionic POD, previously associated with increased levels of lignin and related polyphenols when overexpressed in transgenic tobacco plants (Lagrimini, Plant Physiol. 96 (1991) 577–583), was increased in line C plants. It is suggested that the high IAA level in the transformants, through an induction of ethylene synthesis, increases POD activity and hence also lignin deposition.

Involvement of Indole‐3‐Acetic Acid in Fascicular and Interfascicular Cambial Growth and Interfascicular Extraxylary Fiber Differentiation in Arabidopsis thaliana Inflorescence Stems

The effect of altering the auxin supply on the development and function of the vascular cambium and the differentiation of interfascicular extraxylary fibers was investigated in the inflorescence stem of Arabidopsis thaliana Columbia wild‐type plants. Young, ca. 7‐cm‐tall inflorescence stems were either left untreated (control), decapitated 2 cm above their base and the stump apex treated with indole‐3‐acetic acid (IAA), or ringed 1 cm above their base with N‐1‐naphthylphthalamic acid (NPA), an auxin‐transport inhibitor. At the beginning of the 4‐wk experimental period, the stem base contained vascular bundles with a functioning fascicular cambium; the bundles were separated laterally by a band of immature, interfascicular extraxylary fibers. After ca. 1 wk, stem radial development, as manifested in xylem production by the fascicular cambium and in wall thickening and lignification of interfascicular extraxylary fibers, was finished in control inflorescence stems. Decapitating the inflorescence stem inhibited subsequent stem radial development in the remaining stump. Applying IAA to the stump apex markedly stimulated fascicular xylem production and completed interfascicular extraxylary fiber differentiation. Moreover, IAA application induced the production of interfascicular xylem by initiating interfascicular cambium. Ringing with NPA inhibited stem radial development below the ringing site, while it promoted interfascicular xylem production above. The results indicate that, in the Arabidopsis inflorescence stem, IAA (1) is required for wall thickening and lignification in interfascicular extraxylary fibers and for the initiation of interfascicular cambium and (2) stimulates both fascicular and interfascicular cambial growth.

Seedling shoot, needle and bud development in three provenances of Pinus sylvestris and Pinus contorta cultivated in northern Sweden

The patterns of current‐year shoot, needle and terminal bud elongation in seedlings of three Scots pine (Pinus sylvestris L.) and three lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) provenances were compared during the third and fourth growing seasons after planting. Lodgepole pine produced longer shoots and buds than did Scots pine, mainly because lodgepole pine formed more stem units and elongated at a faster rate. Stem unit length and the duration of shoot and bud elongation differed relatively little between species and provenances. Lammas or polycyclic growth occurred in some lodgepole pine provenances, but not in any Scots pine provenance, and was associated with enhanced shoot elongation. Needle elongation commenced earlier, proceeded at a faster rate, and was greater in lodgepole pine than in Scots pine, but ceased about the same time in all species and provenances. The heat sum required to attain 50% of final length was lower for shoots and needles in lodgepole pine than in Scots ...