Summary: 1. A Study was made of some aspects of the utilization by Spirodela oligorrhiza of ammonium, nitrate, nitride, and organis nitrogenous compounds as sole sources of nitrogen. 2. S. oligorrhiza was grown in axenci culture under carefully defined conditions, and the only factor to be consciously varied were those relating to the nitrogen source. 3. Ammonium, or some product of assimilation, inhibited the utilization of nitrate by inhibiting, at least partially, the uptake of nitrate, and by inhibiting almost completely the reduction of nitrate to nitrite. 4. Nitrite also inhibited the utilization of nitrate. 5. Ammonium and nitrite were taken up and assimilated simultaneously when they were supplied together in the medium. 6. Nitrate reductase and nitrite reductase were found to be adaptive enzymes, being present in S. ologorrhiza only when it was supplied with nitrate or nitrite. There was good correlation between the concentration of nitrate in plants and the level of nitrate reduction that they contained...

The mangrove seedlings Bruguiera and Aviceania develop in a swamp environment where oxygen may be deficient and carbon dioxide in high concentration. Both of these states may be inimical to respiratory processes in plants and especially to the early stages of germination. While vivipary allows the development of the seedlings to a stage where they may better withstand the unfavourable conditions of the environment, nevertheless mangrove seedlings are still liberated when low oxygen concentrations can have severe effects. The developing Avicennia seedling, while on the parent tree, is surrounded by a tough pericarp which restricts gaseous diffusion and induces a partially anaerobic environment around the cotyledon tissues. The detection of high lactic acid levels in Avicennia seedlings may be linked with the carbon dioxide-rich atmospheres which build up around the tissues. It is possible that high levels of carbon dioxide induce the production of lactic acid, a non-toxic product of fermentation, rather than of the more toxic ethanol. Lactic acid is also formed during the initial stages of anaerobiosis in Bruguiera seedlings.

Antirrhinum majus is a model plant used in flower pigmentation studies. Anthocyanin pigment production is mainly controlled by regulation of transcription of the anthocyanin biosynthetic genes. Two types of transcription factors, M Y B and bHLH, together with a WD40 type co-regulator have been shown to regulate the transcription of the anthocyanin biosynthetic genes. In antirrhinum, in addition to the wild type Rosea 1 phenotype, in which pigmentation occurs throughout the inner and outer epidermis of the petal , other complex pigmentation patterns are observed, such as anthocyanins being produced only in the outer (abaxial) epidermis of both lobes and upper tube region of the dorsal petals (roseadorsea phenotype). The major objective of this research project was to understand the genetic regulatory system leading to the development of the two different floral pigmentation patterns in antirrhinum as a means to understanding differential regulation of gene expression in similar cells. Promoter deletion analysis coupled with linker scanning mutagenesis identified the - 1 62 bp to - 1 20 bp region of the Rosea l promoter as i mportant for the regulation of the Rosea l gene. Four putative transcription factor-binding sites within this region : a Wbox, a pyrimidine box, a DOF and a WRKY transcription factor binding site were shown to be important for Rosea l gene regulation. Promoter deletion analysis carried out on the rosea ldorsea promoter showed that the proximal 1 87 bp deletion was, surprisingly, not responsible for the roseadorsea phenotype. Cloning and characterisation of the Rosea l promoter sequence from various Antirrhinum species and accessions verified this finding. The rosealdorsea promoter analysis also indicated that - 1 5 1 bp of the promoter was sufficient for its expression as well as for the maintenance of petal specific expression. The rosea ldorsea allele was also shown to encode a functional protein . In situ hybridisation analysis showed that Rosea l transcripts were present in the inner and outer epidermis of the petal tissue of both wild type and roseadorsea petal tissue. Vascular expression of the Roseal mRNA is indicative of regulation of this gene through sugar or hormonal cues. However, rosea ldorsea transcript levels (in roseadorsea) were much lower than Roseal (wild type). Lowered expression of rosea ldorsea transcripts may be responsible for the overall weak pigmentation in the roseadorsea flowers. Analysis of the intron sequences of the two alleles revealed that many sequence changes were present in the intron 2 of rosea l dorsea. These changes may lead to instability or the lower expression of the rosea l dorsea mRNA and may be responsible for the roseadorsea phenotype. Another possibility is that a fourth Myb gene may be responsible for the roseadorsea phenotype. The role of the Deficiens gene in direct regulation of Rosea l was analysed by RNAi and bioinformatics-based methods. The presence of potential MADS box binding sites in the intron 2 region of the Roseal allele indicated that Rosea l might be directly regulated by Deficiens. Initial experiments using transient assays did not support this suggestion. However, silencing of Deficiens in wild type antirrhinum buds led to the loss of anthocyanin pigments in the petals. Further analysis of the RNAi tissue using SEM revealed that the proper development of conical shaped epidermal cells was also affected . The RNAi tissue also developed chlorophyll pigments underscoring the plasticity of petal identity. This work demonstrated that proper expression of Deficiens is required throughout flowering for anthocyanin pigment production as well as maintenance of petal cell identity. The current investigation revealed that the higher order regulation of the Rosea l alleles in antirrhinum petals is much more complex than initially postulated.

The differential expression of members of the Trifolium repens ACC oxidase (TR-ACO) gene family and accumulation of TR-ACO proteins in white clover roots, and the temporal TR-ACO gene expression and TR-ACO protein accumulation in response to phosphate (Pi) stress has been investigated. Four-node stolon cuttings of wild type and transgenic white clover (designated TR-ACOp::GUS and TR-ACO1p::mGFP5-ER) plants were rooted and acclimatised in Hoagland’s solution, and then subjected to either a Pi sufficiency (1 mM Pi) treatment or a Pi depletion (10 µM Pi) treatment over a designated time course. Using semi quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqRT-PCR) and gene-specific primers it has been determined that the TR-ACO genes are differentially expressed in the roots of white clover. The TR-ACO1 transcript abundance was greater in the lateral roots when compared to the main roots. By immunodetection analysis using antibodies raised against TR-ACO1, recognition of a protein of expected size (ca. 36 kDa) was also greater in the lateral roots. The tissue-specific localisation of TR-ACO1 promoter activity was investigated first by light microscopy using a single genetic line of white clover transformed with a TR-ACO1p::GUS gene construct, and results then confirmed by confocal microscopy using several genetically independent lines of transgenic plants transformed with a TR-ACO1p::mGFP5 ER gene construct. In these lines, the TR-ACO1 promoter activity was primarily located in the meristem of the main and lateral roots, lateral root primordia as well as in the pericycle of the root with nodes of expression in the emerging lateral roots, suggesting a role for ethylene in the development of young tissues where cells are actively dividing. In terms of TR-ACO2, greater transcript abundance and protein accumulation of TR-ACO2 were also observed in the lateral roots when compared to the main roots. Histochemical GUS staining of roots of a single genetically-independent line transformed with a TR-ACO2p::GUS construct showed predominant promoter activity in the mature tissues of both the main and lateral roots but not in the meristematic tissues. In contrast, TR-ACO3 showed greater transcript abundance in the main roots relative to the lateral roots, and the promoter activity, as determined using a single genetically- independent line of TR-ACO3p::GUS transformed plants was predominantly in the mature tissues of the main roots In response to Pi depletion, the members of TR-ACO gene family were temporally expressed in the white clover roots. Using sqRT-PCR, the TR-ACO1 transcript abundance was greater in Pi depleted roots at 12 h and 24 h after Pi depletion in both wild type plants and in the one genetically-independent line of white clover transformed with the TR-ACO1p::mGFP5-ER construct examined. Similarly, by western analysis using both a-TR-ACO1 and commercially available a-GFP antibodies (for the transformed line), a greater accumulation of proteins was consistently observed in Pi depleted roots from the first up to the seventh day after Pi depletion. By confocal microscopy, it was determined for several genetically-independent line of white clover transformed with TR-ACO1p::mGFP5-ER that under Pi depletion more intense GFP fluorescence over a time course of 1 d, 4 d, and 7 d was observed, when compared to plants grown under Pi sufficiency. For TR-ACO2, there was no significant difference in transcript accumulation and protein accumulation in response to short term Pi depletion of up to seven days. However, at 15 d and 21 d after Pi depletion there was a greater protein accumulation in the roots of Pi depleted plants relative to the Pi sufficient roots. Further, when main and lateral roots were compared, a greater protein accumulation occurred in the lateral roots. For TR-ACO3, there was no consistent trend of transcript accumulation in response to Pi depletion over a 24 h period. While a marked reduction in transcript accumulation was noted in Pi depleted roots at 1h, 12 h, 24 h, there was an increase in transcript accumulation at 6 h and 18 h after Pi depletion, indicating that factors other than Pi supply may be affecting gene regulation. Root morphological studies revealed an increase in the main root length and lateral root production in white clover in response to Pi depletion with a greatest growth rate noted between the sixth and ninth day after Pi depletion, and this period overlapped with accumulation of TR-ACO1 protein suggesting a role for ethylene in the Pi stress induced lateral root production in white clover. The differential regulation of the three TR-ACO genes in white clover roots in response to Pi depletion further suggests the divergence in terms of regulation of the ethylene biosynthetic pathway, which may play an important role in fine tuning the responses of plants to particular environmental cues.

Festuca novae-zelandiae (Hack.) Cockayne is an endemic New Zealand perennial tussock forming grass of the family Poaceae. Morphologically F. novae-zelandiae exhibits a number of leaf adaptations associated with dehydration postponement as reflected in the climatic distribution of this species and its occurrence as a physiognomic dominant grass in semi arid short-tussock grasslands. Biochemical studies into the drought tolerance of this species have indicated the occurrence of tissue specific responses with respect to abscisic acid (ABA) and proline accumulation and protein turnover suggestive of a preferential protection of the tiller base and associated meristematic zones at the expense of lamina tissues. Further tissue specific biochemical responses to water-deficit stress in F. novae-zelandiae have been investigated. Changes in water-soluble carbohydrates (WSC) were monitoured over a 49-day dry-down period (decline in soil water content from 30% to 4%) in consecutive leaf segments comprising the leaf base (meristem region), elongation zone, the enclosed and exposed lamina, as well as basal sheath segments from the two next oldest leaves. In fully hydrated leaf tissues polymers of fructose (fructans) were the main WSC present and were mainly low molecular weight fructans of the inulin and neokestose series with the average degree of polymerization (DP) of fructan pools from 6 to 9. The highest fructan concentrations were present towards the leaf base. Fructan concentrations decreased over the course of the dry-down, although remained significantly higher in the meristem region of the tiller base with respect to any other tissue, until tissue water content fell below 45%. By day 49 of the dry-down period, the average DP of the fructan pool in tissues was from 3 to 5. Sucrose content increased in each tissue during the course of the dry-down, and was highest at the leaf base, where a concentration of 200 µmol g-1 dry weight was measured after 49 days of dry-down. The negative correlation between fructan and sucrose content, which indicates an inter-conversion dependent on tissue water content, suggests that, in this specises, fructans serve as a carbohydrate storage pool, while sucrose stabilises the meristem during extreme water deficit.

Repeat purchase of kiwifruit is primarily driven by consumer judgement of internal fruit quality attributes, including those affected by dry matter concentration (DMC) and mineral composition in fruit. This research investigated mechanisms affecting carbohydrate, mineral and water accumulation in 'Hayward' kiwifruit (Actinidia deliciosa), and related these to specific management practices. Canopy manipulation through pruning and treatments such as artificial pollination, defoliation, girdling, thinning and application of the auxin transport inhibitor TIBA, may affect fruit DMC and mineral composition. Leaf photosynthesis and fruit dry matter concentrations (DMC) started to decline as leaf area index values increased above 3-4. In addition to reducing competition for carbohydrates between vegetative and reproductive growth, leader pruning probably increased DMCs of fruit in the leader zone by improving light interception. Photosynthesis was not affected by crop loads between 20- 60 fruit m-2, but was consistently higher on non-terminating (long) shoots than on terminating (short) shoots, as were fruit DMCs. Differences in photosynthetic rate of leaves on these two shoot types were attributed to differences in shoot exposure to the sun, and also to the greater demand for carbohydrate within long shoots. Leaves subtending fruit may increase Ca, and to a lesser extent Mg, flow into fruit, however their accumulation was not affected by leaves outside the fruiting shoot. Xylem sap Ca and Mg concentrations were higher in shoots with a high rather than a low leaf: fruit (L:F) ratio and this may, at least partially, relate to the increase in shoot transpiration that occurs as shoot L:F ratios increase. Within vine variation in fruit Ca concentrations may reflect variations in xylem sap flow rates and Ca concentrations of xylem sap reaching fruit. Calcium translocation may occur independently of ion movement in the transpiration stream. Timing and extent of vascular differentiation in flower and fruitlet pedicels, possibly regulated by auxin, may influence fruit Ca accumulation. It is likely that early differentiation of vascular tissue in flower and fruitlet pedicels influenced cell division and subsequent (carbohydrate) sink strength of fruit by determining availability of carbohydrate for partitioning into cell walls. While growers have the potential to induce minor changes in fruit DMC, further increases will depend on the separation of carbohydrate and water accumulation. Further research is required to elucidate the mechanisms regulating phloem transport and unloading of sucrose in kiwifruit.

Mr Albert investigated the genetic mechanisms controlling complex floral and vegetative pigmentation patterns in Petunia. He discovered two new MYB transcription factors that control the timing and spatial location of anthocyanin pigment production in flowers and leaves, giving rise to specific colour patterns. He showed that complex pigmentation patterns are formed by tightly controlling the expression of genes required to synthesise anthocyanin pigments and involves proteins that both activate genes and repress them from being expressed. The interactions between distinct classes of transcription factors form an intricate network and hierarchy, allowing fine control of gene expression and strict control of pigment production. These findings will aid in the development of ornamental plants with new pigmentation patterns and also this research serves as a model for how plants control the expression of genes to produce health-promoting plant compounds.

Mr Albert investigated the genetic mechanisms controlling complex floral and vegetative pigmentation patterns in Petunia. He discovered two new MYB transcription factors that control the timing and spatial location of anthocyanin pigment production in flowers and leaves, giving rise to specific colour patterns. He showed that complex pigmentation patterns are formed by tightly controlling the expression of genes required to synthesise anthocyanin pigments and involves proteins that both activate genes and repress them from being expressed. The interactions between distinct classes of transcription factors form an intricate network and hierarchy, allowing fine control of gene expression and strict control of pigment production. These findings will aid in the development of ornamental plants with new pigmentation patterns and also this research serves as a model for how plants control the expression of genes to produce health-promoting plant compounds.

Mr Albert investigated the genetic mechanisms controlling complex floral and vegetative pigmentation patterns in Petunia. He discovered two new MYB transcription factors that control the timing and spatial location of anthocyanin pigment production in flowers and leaves, giving rise to specific colour patterns. He showed that complex pigmentation patterns are formed by tightly controlling the expression of genes required to synthesise anthocyanin pigments and involves proteins that both activate genes and repress them from being expressed. The interactions between distinct classes of transcription factors form an intricate network and hierarchy, allowing fine control of gene expression and strict control of pigment production. These findings will aid in the development of ornamental plants with new pigmentation patterns and also this research serves as a model for how plants control the expression of genes to produce health-promoting plant compounds.

Mr Albert investigated the genetic mechanisms controlling complex floral and vegetative pigmentation patterns in Petunia. He discovered two new MYB transcription factors that control the timing and spatial location of anthocyanin pigment production in flowers and leaves, giving rise to specific colour patterns. He showed that complex pigmentation patterns are formed by tightly controlling the expression of genes required to synthesise anthocyanin pigments and involves proteins that both activate genes and repress them from being expressed. The interactions between distinct classes of transcription factors form an intricate network and hierarchy, allowing fine control of gene expression and strict control of pigment production. These findings will aid in the development of ornamental plants with new pigmentation patterns and also this research serves as a model for how plants control the expression of genes to produce health-promoting plant compounds.