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Within two years of their discovery in 1977, introns were found to have a positive effect on gene expression. Numerous examples of stimulatory introns have been described since then in very diverse organisms, including plants. In some cases, the mechanism through which the intron affects expression is readily understood. However, many introns that affect expression increase mRNA accumulation through an unknown mechanism, referred to as intron-mediated enhancement (IME). Despite several decades of research into IME, and the clear benefits of using introns to increase transgene expression, little progress has been made in understanding the mechanism of IME. Several fundamental questions regarding the role of transcription and splicing, the sequences responsible for IME, the involvement of other factors, and the relationship between introns and promoters remain unanswered. The more we learn about the properties of stimulating introns, the clearer it becomes that the effects of introns are unfamiliar and difficult to reconcile with conventional views of how transcription is controlled. We hypothesize that introns increase transcript initiation upstream of themselves by creating a localized region of accessible chromatin. Introns might represent a novel kind of downstream regulatory element for genes transcribed by RNA polymerase II.

To more precisely define the positions from which certain intronic regulatory sequences increase mRNA accumulation, the effect of a UBIQUITIN intron on gene expression was tested from six different positions surrounding the transcription start site (TSS) of a reporter gene fusion in Arabidopsis thaliana The intron increased expression from all transcribed positions but had no effect when upstream of the 5'-most TSS. While this implies that the intron must be transcribed to increase expression, the TSS changed when the intron was located in the 5'-untranslated region (UTR), suggesting that the intron affects transcription initiation. Remarkably, deleting 303 nucleotides of the promoter including all known TSSs and all but 18 nucleotides of the 5'-UTR had virtually no effect on the level of gene expression as long as an intron containing stimulatory sequences was included. Instead, transcription was initiated in normally untranscribed sequences the same distance upstream of the intron as when the promoter was intact. These results suggest that certain intronic DNA sequences play unexpectedly large roles in directing transcription initiation and constitute a previously unrecognized type of downstream regulatory element for genes transcribed by RNA polymerase II.

Introns are often added to transgenes to increase expression, although the mechanism through which introns stimulate gene expression in plants and other eukaryotes remains mysterious. While introns vary in their effect on expression, it is unknown whether different genes respond similarly to the same stimulatory intron. Furthermore, the degree to which gene regulation is preserved when expression is increased by an intron has not been thoroughly investigated. To test the effects of the same intron on the expression of a range of genes, GUS translational fusions were constructed using the promoters of eight Arabidopsis genes whose expression was reported to be constitutive (GAE1, CNGC2 and ROP10), tissue specific (ADL1A, YAB3 and AtAMT2) or regulated by light (ULI3 and MSBP1). For each gene, a fusion containing the first intron from the UBQ10 gene was compared to fusions containing the gene's endogenous first intron (if the gene has one) or no intron. In every case, the UBQ10 intron increased expression relative to the intronless control, although the magnitude of the change and the level of expression varied. The UBQ10 intron also changed the expression patterns of the CNGC2 and YAB3 fusions to include strong activity in roots, indicating that tissue specificity was disrupted by this intron. In contrast, the regulation of the ULI3 and MSBP1 genes by light was preserved when their expression was stimulated by the intron. These findings have important implications for biotechnology applications in which a high level of transgene expression in only certain tissues is desired.

Related motifs strongly increase gene expression when added to an intron located in coding sequences. Many introns greatly increase gene expression through a mechanism that remains elusive. An obstacle to understanding intron-mediated enhancement (IME) has been the difficulty of locating the specific intron sequences responsible for boosting expression because they are redundant, dispersed, and degenerate. Previously we used the IMEter algorithm in two independent ways to identify two motifs (CGATT and TTNGATYTG) that are candidates for involvement in IME in Arabidopsis. Here we show that both motifs are sufficient to increase expression. An intron that has little influence on expression was converted into one that increased mRNA accumulation 24-fold and reporter enzyme activity 40-fold relative to the intronless control by introducing 11 copies of the more active TTNGATYTG motif. This degree of stimulation is twice as large as that of the strongest of 15 natural introns previously tested in the same reporter gene. Even though the CGATT and TTNGATYTG motifs each increased expression, and CGATT matches the NGATY core of the longer motif, combining the motifs to make TTCGATTTG reduced the stimulating ability of the TTNGATYTG motif. Additional substitutions were used to test the contribution to IME of other residues in the TTNGATYTG motif. The verification that these motifs are active in IME will improve our ability to predict the stimulating ability of introns, to engineer any intron to increase expression to a desired level, and to explore the mechanism of IME by seeking factors that might interact with these sequences.

Introns that elevate mRNA accumulation have been found in a wide range of eukaryotes. However, not all introns affect gene expression, and direct testing is currently the only way to identify stimulatory introns. Our genome-wide analysis in Arabidopsis thaliana revealed that promoter-proximal introns as a group are compositionally distinct from distal introns and that the degree to which an individual intron matches the promoter-proximal intron profile is a strong predictor of its ability to increase expression. We found that the sequences responsible for elevating expression are dispersed throughout an enhancing intron, as is a candidate motif that is overrepresented in first introns and whose occurrence in tested introns is proportional to its effect on expression. The signals responsible for intron-mediated enhancement are apparently conserved between Arabidopsis and rice (Oryza sativa) despite the large evolutionary distance separating these plants.

The extensive mechanistic and regulatory interconnections between the various events of mRNA biogenesis are now recognized as a fundamental principle of eukaryotic gene expression, yet the specific details of the coupling between the various steps of mRNA biogenesis do differ, and sometimes dramatically, between the different kingdoms. In this review, we emphasize examples where plants must differ in this respect from other eukaryotes, and highlight a recurring trend of recruiting the conserved, versatile functional modules, which have evolved to support the general mRNA biogenesis reactions, for plant-specific functions. We also argue that elucidating the inner workings of the plant 'mRNA factory' is essential for accomplishing the ambitious goal of building the 'virtual plant'.

Introns are often required for full expression of genes in organisms as diverse as plants, insects, nematodes, yeast, and mammals. To explore the potential mechanisms of intron-mediated enhancement in Arabidopsis thaliana, the effect of varying the position of an intron was determined using a series of reporter gene fusions between TRYPTOPHAN BIOSYNTHESIS1 (TRP1) and GUS. Two introns that differ in the degree to which they stimulate expression were individually tested at six locations within coding sequences and two positions in the 3'-UTR. The ability of the first introns from both the TRP1 and POLYUBIQUITIN10 (UBQ10) genes to elevate mRNA accumulation in transgenic plants was found to decline with distance from the promoter, despite their being efficiently spliced from all coding sequence locations. Neither intron significantly enhanced mRNA accumulation when positioned 1.1 kb or more from the start of transcription. In addition, measurements of GUS enzyme activity revealed that both introns at all locations elevated GUS activity more than they enhanced mRNA accumulation. The stimulation mediated by two of four other introns tested at the position nearest the promoter was also greater at the level of GUS activity than mRNA accumulation. These findings support a model in which introns increase transcription and promote translation by two distinct mechanisms.

The composition of humid, unfertilised, short‐tussock (Festuca‐Rytidosperma‐Poa) grasslands was recorded on 42 permanent transects in 1959, 1970–72, and 1995–96 in the Wairau catchment, Marlborough. Changes in frequency of the most abundant plant species and bare ground were analysed. Reduced levels of grazing, browsing, and fire prompted significant recovery of native shrubs, tall tussocks (Chionochloa), and herbs. Invasive exotics (Hieracium, Anthoxanthum, Agrostis) also increased. Short native grasses (Poa, Elymus), a native herb (Acaena spp.), and an exotic herb (Rumex acetosella) declined. Hieracium pilosella, H. caespitosum, and H. lepidulum were rare or absent in 1959. By 1996 they had established on half the transects, but were still in the early stages of invasion (median frequencies ≤ 5%). There was no correlation between the increase in Hieracium and decline in other species, suggesting that other factors were driving the compositional changes. However, Hieracium species can establish ...

To investigate how plants acquire and assimilate sulfur from their environment, we isolated and characterized two mutants of Arabidopsis thaliana deficient in sulfate transport. The mutants are resistant to selenate, a toxic analogue of sulfate. They are allelic to each other and to the previously isolated sel1 (selenate-resistant) mutants, and have been designated sel1-8 and sel1-9. Root elongation in these mutants is less sensitive to selenate than in wild-type plants. Sulfate uptake into the roots is impaired in the mutants under both sulfur-sufficient and sulfur-deficient conditions, but transport of sulfate to the shoot is not affected. The sel1 mutants contain lesions in the sulfate transporter gene Sultr1;2 located on the lower arm of chromosome 1. The sel1-1, sel1-3 and sel1-8 mutants contain point mutations in the coding sequences of Sultr1;2, while the sel1-9 mutant has a T-DNA insertion in the Sultr1;2 promoter. The Sultr1;2 cDNA derived from wild-type plants is able to complement Saccharomyces cerevisiae mutants defective in sulfate transport, but the Sultr1;2 cDNA from sel1-8 is not. The Sultr1;2 gene is expressed mainly in roots, and accumulation of transcripts increases during sulfate deprivation. Examination of transgenic plants containing the Sultr1;2 promoter fused to the GUS-reporter gene indicates that Sultr1;2 is expressed mainly in the root cortex, the root tip and lateral roots. Weaker expression of the reporter gene was observed in hydathodes, guard cells and auxiliary buds of leaves, and in anthers and the basal parts of flowers. The results indicate that Sultr1;2 is primarily involved in importing sulfate from the environment into the root.

The distribution and density of Hieracium pilosella, H. caespitosum, and H. lepi‐dulum seedlings were quantified at an early stage of invasion of a tall‐tussock (Chionochloa) and a more open short‐tussock (Festuca‐Poa) grassland. Seedling and available (unoccupied) microsites were compared in 11 stands. Recorded variables included establishment substrate, overhanging cover, and proximity and height of neighbouring plants. Total Hieracium cover was

Many introns significantly increase gene expression through a process termed intron-mediated enhancement (IME). Introns exist in the transcribed DNA and the nascent RNA, and could affect expression from either location. To determine which is more relevant to IME, hybrid introns were constructed that contain sequences from stimulating Arabidopsis thaliana introns either in their normal orientation or as the reverse complement. Both ends of each intron are from the non-stimulatory COR15a intron in their normal orientation to allow splicing. The inversions create major alterations to the sequence of the transcribed RNA with relatively minor changes to the DNA structure. Introns containing portions of either the UBQ10 or ATPK1 intron increased expression to a similar degree regardless of orientation. Also, computational predictions of IME improve when both intron strands are considered. These findings are more consistent with models of IME that act at the level of DNA rather than RNA.

Tree size and age structures, treefall and canopy gap characteristics, and regeneration responses to treefalls were compared for three stands of old-growth beech (Notho- fagus) forest dominated by N. fusca and N. menziesii on the South Island, New Zealand. Treefall gaps (up to 1000 m2) were most often caused by standing trees killed by drought and/or insect attack, or by trees snapped by wind. The causes of gap formation and the size and age distributions of treefall gaps varied between localities because of spatial and temporal differences in the histories of disturbance. At Fergies Bush where drought-related dieback had produced many large gaps with standing dead trees, gaps were generally young. Con- versely, at Station Creek, small, old gaps formed by bolesnap dominated the disturbance regime. At Rough Creek, gaps of all ages and sizes were found along with an almost complete fern cover, and abundant shrubs and occasional subcanopy hardwood trees. Although overall patterns of regeneration were unrelated to differences in gap size, the relative abundance of N. fusca and N. menziesii varied between localities according to the seemingly minor differences in forest structure and disturb- ance history described above. Interpretations of regeneration response to gap parameters, therefore, need to account for differences in disturbance history between sites. Differences in the disturbance history between localities will also influ- ence rates of gap closure, and because closure rates are used to estimate forest turnover times, meaningful comparisons of disturbance regimes for different forest types can only be made if this intersite variability is addressed.

Size and age structure analysis, dated past disturbances, treefall replacement patterns, and spatial pattern analysis were used to reconstruct the developmental history of two old-growth Nothofagus fusca/N. menziesii stands, South Island, New Zealand. Diameter and height class distributions suggested that N. menziesii was replacing N. fusca, however, stand history reconstruction analysis showed that both species had regenerated intermittently after small-scale disturbances. Although large-scale disturbances such as blowdowns may occasionally generate even-aged stands, gap-phase regeneration maintains the forests in compositional equilibrium. In the absence of other competing tree species and understorey plants the two species appear to coexist by way of different life history strategies, where one species (N. menziesii) has low juvenile mortality and the other (N. fusca) has faster height growth rates and greater longevity and adult survivorship.

Summary The expression of the Arabidopsis thaliana PAT1 gene, which encodes the tryptophan biosynthetic enzyme phosphoribosylanthranilate transferase, was investigated using translational fusions of the PAT1 promoter to the GUS reporter gene. Independent stably transformed A. thaliana lines containing a single copy of a fusion that includes the entire plastid transit peptide and the first two introns of PAT1 had on average 30 times more GUS enzyme activity than plants transformed with a construct in which GUS was fused a short distance downstream of the PAT1 start codon. Plants containing the construct without introns or leader peptide accumulated undetectable amounts of PAT1-GUS fusion protein and mRNA, even though the transcriptional rate of both fusion constructs was comparable. Fusions containing the entire transit peptide and either of the first two introns yield as much GUS activity as constructs containing both introns, but constructs containing the transit peptide but no introns give rise to much lower levels. Therefore, introns greatly enhance the expression of PAT1-GUS fusions, and they act post-transcriptionally to increase the steady-state level of mRNA.

An Arabidopsis thaliana gene encoding phosphoribosylanthranilate transferase is shown to be the gene that is defective in blue fluorescent trp1 mutant plants. This gene, named PAT1, was isolated using an A. thaliana cDNA clone that suppressed an Escherichia coli trpD(-) mutation. The PAT1 coding region is homologous to those for the phosphoribosylanthranilate transferases from many microorganisms. Unlike other genes involved in aromatic amino acid biosynthesis in A. thaliana, PAT1 appears to be a single-copy gene. PAT1 was demonstrated to be the gene that is defective in blue fluorescent trp1 mutants by two methods: genetic complementation in transgenic plants and genetic mapping studies. This is the first report of cloning a plant phosphoribosylanthranilate transferase gene. The PAT1 gene should prove useful as a selectable marker for transformation or a visible reporter of gene expression when used in conjunction with trp1 plants.

Age-states, population structure, and seedling regeneration of the grass Chionochloa pallens were in- vestigated in eight alpine grassland stands representing the range of canopy cover (ca. 5 - 75 %) and basal area (112 - 3889 m2/ha-1) in the headwaters of the Avoca catchment, Canterbury, New Zealand. In open-canopy stands (< 50 % canopy cover, < 2000 m2/ha-1 basal area) C. pallens individuals were clearly distinguishable. Rela- tionships between basal diameter, numberof tillers, height, and % crown death suggested four putative age-states: 'seedlings', 'juvenile tussocks', 'mature tussocks' and 'senescent tussocks'. Four types of population structure were identified depending on the proportions of seedlings and juvenile and mature tussocks. C. pallens seedlings were present in seven stands at densities ranging from 0.6 to 123.4 /m2. They were most frequent on microsites pro- tected from frost-heave by short herbaceous vegetation or litter, but with little competition from taller vegetation. Differences in seedling densities between stands partly reflected the proximity and abundance of seed sources and degree of protection from frost-heave. C. pallens population structures appear to be influenced by the fre- quency and magnitude of geomorphic disturbances and browsing by introduced animals.

Size and age structure analysis were used to reconstruct the population dynamics of six apparently old and stable stands dominated by the conifers Libocedrus bidwillii and/or Dacrydium biforme in Camp Creek, West land, New Zealand. All stands contained relatively even-aged groups of Libocedrus that had probably originat ed after either mass movement or windthrow. The age distributions of stands reflected the effects of distur bances 200-400 yr ago. There was no evidence to support synchronous failure of regeneration of Libocedrus and Dacrydium bi forme. Between 1600 and 1800 A.D., the period of the postulated conifer regeneration gap, over 70% of trees of these species had established. A lack of Libocedrus regeneration over the last 200 yr reflects a lack of distur bance over that time. Because of the dominating influence of disturbances and other factors it may be inap propriate to use tree population structures alone to substantiate or refute claims of recent climate change.