A cDNA clone encoding Brassica calmodulin

A 834 bp cDNA encoding calmodulin (CaM) has been isolated from Brassica juncea. On Northern analysis this cDNA hybridises this cDNA to mRNAs of about 0.9 kb in leaf, silique and peduncle. Genomic Southern analysis indicates the presence of a CaM multigene family in Brassica juncea. Comparison of the predicted amino acid sequence of Brassica CaM with that of Arabidopsis CaM ACaM-2 and ACaM-3 showed 100% homology, which is not unusual, since both plants belong to the family Cruciferae. In situ hybridisation studies on Brassica seedlings using a digoxigenin-labelled RNA probe showed that high levels of CaM mRNA were detected in the leaf primordia and the shoot apical meristem, and to a lesser degree, in the zone of root elongation of the root tip. The occurrence of a higher rate of cell division and growth in these regions than its surrounding tissue may possibly be related to higher levels of CaM mRNA.     

Definition of constitutive gene expression in plants: the translation initiation factor 4A gene as a model

The NeIF-4A10 gene belongs to a family of at least ten genes, all of which encode closely related isoforms of translation initiation factor 4A. The promoter region of NeIF-4A10 was sequenced, and four mRNA 5 ends were determined. Deletions containing 2750, 689 and 188 bp of untranscribed upstream DNA were fused to the GUS reporter gene and introduced into transgenic tobacco. The three constructs mediated GUS expression in all cells of the leaf, stem and shoot apical meristem. Control experiments using in situ hybridization and tissue printing indicated that the observed GUS expression matches the expression patterns of NeIF-4A mRNA and protein. This detailed analysis at the level of mRNA, protein and reporter gene expression shows that NeIF-4A10 is an ideal constitutively expressed control gene. We argue that inclusion of such a control gene in experiments dealing with specifically expressed genes is in many cases essential for the correct interpretation of observed expression patterns.     

Phalaenopsis bellina (Rchb.f.) Christenson,a segregate from P. violacea Witte (Orchidaceae: Aeridinae)

A reevaluation of floral and vegetative morphology together with data from analysis of floral fragrance and flavonoid co-pigment chemistry suggest that specific status is warranted for the two kinds ofPhalaenopsis violacea Witte known informally as the “Bornean type” and the “Malayan type.”Phalaenopsis violacea var.bellina Rchb.f. is elevated to specific rank, providing a name for the “Bornean type” plants.     

β-glucuronidase as a marker for clonal analysis of tomato lateral roots

In higher plants, the root-shoot axis established during embryogenesis is extended and modified by the development of primary and lateral apical meristems. While the structure of several shoot apical meristems has been deduced by combining histological studies with clonal analysis, the application of this approach to root apical meristems has been limited by a lack of visible genetic markers. We have tested the feasibility of using a synthetic gene consisting of the maize transposable elementActivator (Ac) inserted between a 35S CaMV promoter and the coding region of a -glucuronidase (GUS) reporter gene as a means of marking cell lineages in roots. The GUS gene was activated in individual cells byAc excision, and the resulting sectors of GUS-expressing cells were detected with the histochemical stain X-Gluc. Sectors in lateral roots originated from bothAc excision in meristematic cells and from parent root sectors that bisect the founder cell population for the lateral root initial. Analysis of root tip sectors confirmed that the root cap, and root proper have separate initials. Large sectors in the body of the lateral root encompassed both cortex and vascular tissues. The number of primary initial cells predicted from the size and arrangement of the sectors observed ranged from two to four and appeared to vary between roots. We conclude that transposon-based clonal analysis using GUS expression as a genetic marker is an effective approach for deducing the functional organization of root apical meristems.     

Floral development of Bougainvillea spectabilis Willd., Boerhaavia diffusa L. and Mirabilis jalapa L. (Nyctaginaceae)

Three morphological problems were investigated in three species of the Nyctaginaceae: epiphylly, phyllotaxis and placentation. Epiphylly, which occurs in Bougainvillea spectabilis , is the result of ontogenetic displacement resulting from the activity of an intercalary meristem at the base of the floral bract and the floral bud. Floral development of Bougainvillea spectabilis was compared with that of Boerhaavia diffusa and Mirabilis jalapa . Considerable variation occurs with regard to the number and arrangement of stamens. Five stamens are initiated simultaneously, alternate to the petals, in Mirabilis . In Bougainvillea , eight stamens arise sequentially at divergence angles suggestive of a 3/8 spiral. No developmental evidence was found to support the derivation of the eight stamens from a two whorled pentamerous androecium. Boerhaavia normally has only two stamens which most frequently are initiated toward opposite sides of the floral apex, but may also be formed in a 2/5 to 3/8 divergence. In some flowers only one or three stamens are formed. The gynoecium is formed in the same way in all three species: growth occurs in a crescent-shaped zone at the periphery of the floral apex thus producing the gynoecial wall. The single ovule, which is basal in the mature gynoecium, is formed from the gradual upgrowth and transformation of the floral apex and is developmentally terminal. Even the two-layered tunica is maintained as the floral apex is transformed into the ovule primordium. If 'carpel' is defined traditionally as a folded megasporophyll which bears and encloses ovule(s) then carpels are not present in the gynoecia of the three species studied. If 'carpel' is re-defined as an appendage which encloses ovule(s), then the gynoecia of the Nyctaginaceae are carpellate. However, the ovules remain cauline regardless of which definition is adopted.     

Developmental study onHypolepis punctata (Thunb.) Mett.

The third petiolar bud ofHypolepis punctata appears on the basiscopic lateral side of the petiole above the fairly developed first petiolar bud. This investigation clarified the fact that the third bud is formed neither by the activity of the meristem of the first bud nor by the meristem directly detached from the shoot apical meristem, but is initiated in the cells involved in the abaxial basal part of the elevated portion of the leaf primordium. Thus the third bud is of phyllogenous origin. This investigation further revealed that the cells to initiate the third bud are originally located in the abaxial side of the leaf apical cell complex like the cells to initiate the first bud, but are not incorporated into the meristem of the first. After the first, second and third petiolar buds have been initiated, they are carried up into fairly high regions on the petiolar base by the intercalary growth which occurs in the leaf base below the insertion level of the first and the second buds.     

Reproductive ecology of dioecious Siparuna (Monimiaceae) in Ecuador–a case of gall midge pollination

The reproductive ecology of the dioecious genus Siparuna , belonging to the primitive family Monimiaceae, was investigated in Ecuador. Thirteen species were studied, and species growing at high altitudes (above 500 m) were compared with species growing at lower altitudes. The genus has specialized flowers pollinated by Cecidomyiidae (Asynapta sp. and other genera) that lay eggs chiefly in male flowers since these are more easily accessible than female flowers due to their morphology. Several traits ensure that pollen transfer is accomplished: 1. long flowering of individuals and single flowers; 2. specific floral morphology (unisexuality, landing platform, restricted pore) allowing the flowers to be visited by Cecidomyiidae seeking egg-laying sites, and 3. a low number of ovules that have to be pollinated in each flower. The low number of ovules per flower results in a high pollen-ovule (P/O) ratio even though pollen production in male flowers is only moderate.
High altitude species had larger flowers, higher pollen-ovule ratios, fewer ovules per female flower, and longer periods of anthesis. Gender differences were seen in the longer functionality of female flowers; the larger numbers of male flowers produced; and the average diameter at breast height (DBH) biased for one of the two sexes depending on the species investigated, with no clear trend in the size distributions of individuals of both sexes. Sex ratios departed significantly from unity both to male and female bias but usually were in favour of males. There were no general differences in the ratio of functional male to female flowers at the time of observation nor in the sizes of male and female flowers. The evolution of unisexual flowers in Siparuna can be explained as a result of the differential predation by larvae: unimportant in male flowers, destructive if occurring in female flowers.     

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 10⁶ clones from as little as 1 μg of total RNA.