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Physical properties and microstructure of hybrid processed cheeses formulated with plant protein and milk protein ingredients
(Elsevier Ltd, 2026-02-01) Lu D; Roy D; Acevedo-Fani A; Singh H; Waterland M; Ye A
Hybrid processed cheese analogues (HPCAs) containing either mung bean (MPI) or hemp protein (HPI) with rennet casein (RC) at various ratios were prepared and analysed to understand their spatial and microstructural distribution and related physical properties, such as rheological properties, texture profile, meltability, and stretchability. In addition, protein composition and secondary protein structure were studied using SDS–PAGE and FTIR spectroscopy, while CLSM and TEM were employed to visualise the microstructure of the cheese matrix. Results indicated that plant protein types and concentration significantly affected the physical properties and microstructure of HPCAs. The addition of 30 % or more plant protein altered the physical and textural properties as well as the microstructure of the cheese analogues, with a decrease in β-sheet content and an increase in random coil structures. Mung bean protein–based HPCAs exhibit greater stretchability (e.g. 93.8 mm in 30 % MPI vs 41.53 mm in 30 % HPI), rheological, and textural properties, but not meltability (e.g. 1 % in 70 % MPI vs 48 % in 70 % HPI), compared with the hemp protein system at the same mixing ratios. This difference can be attributed to the size of the plant protein aggregation. All data were analysed by one-way ANOVA with Tukey's test (p < 0.05). These findings deepen our understanding of plant protein-based and hybrid cheeses, paving the way for optimised plant-based dairy alternatives.
Fabrication, characterisation, and application of functional protein aggregates derived from faba bean protein isolates : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Auckland, New Zealand
(Massey University, 2025-07-14) Hu, Yinxuan
This thesis explores the preparation, characterisation, and applications of plant protein aggregates, derived from faba bean protein isolate (FPI). The formation of FPI aggregates was accomplished by various methods, including pH adjustments, salt addition, heat treatment, sonication, and thermosonication (TS). The physico-chemical properties and technofunctional characteristics of FPI aggregates formed by different treatments, such as ζ-potential, solubility, emulsification capability, and particle sizes, were also characterised in this study. Furthermore, the microstructure of the FPI aggregates in solutions was examined using various techniques, including light scattering, microscopies (TEM and SEM), and small angle neutron scattering. Additionally, this project further developed the TS method for formation of FPI fibrillar aggregates at pH 2 and amorphous aggregates at pH 7. The characteristics of FPI aggregates formed by TS and conventional heat treatment (CH) were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography linked to tandem mass spectrometry (LC-MS/MS). In addition, Thioflavin T (ThT) fluorescence, Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD) were applied to investigate the differences in secondary structure between CH-treated FPI and TS-treated FPI, indicating that TS effectively converted FPI structures to be enriched in β-sheets. The gelation behaviours of different FPI aggregates at 10 wt% were studied by examining their rheological properties and observing the microstructure using scanning electron microscopy (SEM), indicating that TS-treatment of FPI at pH 7 facilitated the formation of stronger protein hydrogels. The functionality of FPI aggregates fabricated from various treatments at the oil-water and oil-air-water interfaces was also characterised. Emulsions (O/W) with various oil factions (ϕ) ranging from 0.2 (dilute emulsions) to 0.75 (high internal phase emulsions, HIPEs), were stabilised by suitable FPI aggregates selected based on their different physico-chemical properties. The findings indicate that higher FPI concentrations (~5 wt%) and pH values (~pH 9) result in better emulsification capabilities. Among all FPI aggregates studied in this project, fibrillar aggregates exhibited the best emulsification performance as they could stabilise emulsions with oil content up to 75% (v/v). However, emulsions stabilised by FPI aggregates induced from TS at pH 7 had the greatest application potential due to their long-term stability (up to 28 days) and compatibility with a neutral pH environment. Therefore, another study in this thesis was to investigate the application of FPI aggregates in stabilising vegetable oil-based whipped creams. TS-treated FPI at pH 7 exhibited superior functional properties compared to other treatments, such as CH and ultrasonication (US), in terms of visual appearance, overrun, and stability of whipped cream. Overall, this project provides fundamental insights into the physical-chemical and techno-functional properties of FPI aggregates, including their ability to stabilise and form emulsions, gels, and foams, with an emphasis on their potential applications in innovative food products such as 3D-printed emulsion gels and plant based whipped cream. The enhanced physicochemical and techno-functional properties of FPI aggregates fabricated in this study showed a great application potential as novel food ingredients for formulation of plant-based food products.
Alternative proteins vs animal proteins: The influence of structure and processing on their gastro-small intestinal digestion
(Elsevier Ltd, 2022-04) Kaur L; Mao B; Beniwal AS; Abhilasha; Kaur R; Chian FM; Singh J
Background: Digestibility, an indicator of protein bioavailability, is essentially a measure of the susceptibility of a protein towards proteolysis. Proteins with higher digestibility have been linked with better health outcomes. Animal proteins are generally considered to be of better nutritional value than plant proteins not only because they are a good source of essential amino acids but also due to their higher digestibility in the human gastro-intestinal tract. With the recent emergence of alternative food protein sources, which are now processed in a completely new way to design new foods or new versions of the conventional foods, it has become extremely important to understand their digestion characteristics. Scope and approach: This review discusses the factors that affect protein digestibility, including protein source, structure, type of processing, and modification, with a particular focus on the effects of non-protein components present in food matrix. Key findings and conclusions: To obtain the desired functionality, particularly for alternate proteins, numerous physical, chemical, and enzymatic methods for modification have been reported. These modifications may alter structural characteristics of proteins by inducing structural modifications such as protein unfolding, crosslinking, and aggregation. Depending upon the protein reactivity during processing, the susceptibility of proteins towards hydrolysis by digestive enzymes might change, affecting not only the overall protein digestibility but also the rates of release of polypeptides and amino acids. The faster rates of protein digestion have been linked with muscle anabolism, suggesting the need and importance of classifying the new, emerging and alternative protein sources according to their rates of digestion into rapidly (RDP), slowly digestible (SDP) and resistant (RP) proteins. More research needs to be focussed on converting, through processing, the undigestible or RP into RDP or SDP to achieve better health outcomes.
The development of extruded meat alternatives using Maillard-reacted beef bone hydrolysate and plant proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
(Massey University, 2020) Chiang, Jie Hong
This research thesis aimed to process beef bone extract into a flavoursome protein ingredient to be added to extruded meat analogues to form meat alternatives and study their impact on the structural, textural, and sensory properties of meat alternatives. The thesis consists of three main parts. In the first part, two methods namely enzymatic hydrolysis and Maillard reaction (MR) treatments were evaluated for their suitability of modifying the flavour character of beef bone extract to become flavoursome protein ingredients. The second part studied the effects of soy protein concentrate (SPC) to wheat gluten (WG) ratio as a way of improving the structural and textural properties of current extruded meat analogues. The third part studied the effects of flavoursome protein ingredient (i.e. Maillard-reacted beef bone hydrolysate) with plant proteins on extruded meat alternatives. It also investigated the effects of moisture contents on extruded meat alternatives and their application in sausages. To begin, an experimental study on the effects of enzymatic hydrolysis treatments (i.e. single, simultaneous and sequential) on the physicochemical properties of beef bone extract using Protamex®, bromelain, and Flavourzyme® was conducted. Next, the changes in the physicochemical properties and volatile compounds of beef bone hydrolysates during heat treatment as a result of the MR were investigated. Beef bone hydrolysates were combined with ribose in aqueous solutions and heated at 113°C to produce Maillard reaction products (MRPs). Results showed that Flavourzyme® was the most effective in increasing the proportion of low Mw peptides, reducing viscosity and enhancing the flavour intensity of beef bone extract. Concurrently, the effects of SPC to WG ratio at a constant mass of SPC and WG on the physicochemical properties of extruded meat analogues were studied. Meat analogues containing 30%WG showed the highest degree of texturisation, fibrous structure, hardness and chewiness using instrumental and sensory analysis. For the third part of this research thesis, the effects of flavoursome protein ingredient (i.e. Flavourzyme®-MRP) at different concentrations (0, 10, 20, 30 and 40% wet weight) with plant proteins on extruded meat alternatives were investigated. Meat alternatives containing 20%MRP obtained the highest sensory scores for appearance, meaty aroma, meaty taste, and overall acceptability. Results showed that the addition of MRP with soy protein concentrate and wheat gluten to produce meat alternatives changed the textural, structural, and sensory properties significantly. The effects of moisture content (MC) on the physicochemical properties of extruded meat alternatives made from Flavourzyme®-MRP and plant proteins were studied. Samples were extruded at different dry feed rate of 1.8, 2.2, 2.6 and 3.0 kg/h to obtain MC of 60%MC, 56%MC, 52%MC and 49%MC, respectively. Meat alternatives at 49%MC were the closest in terms of both textural and microstructural properties to reference sample, boiled chicken breast. Results showed that the change in MC as a process parameter played an important role in the formation of fibrous structure in extruded meat alternatives. Lastly, the physicochemical properties of sausages made from extruded meat alternatives at different MC were conducted. Five sausages made from meat alternatives (S49%MC, S52%MC, S56%MC and S60%MC) and chicken breast (SCB) as a reference sample were prepared. Results showed that S49%MC had the highest sensory scores among all sausages made from meat alternatives. However, SCB obtained the highest sensory scores for all attributes except for appearance among all sausages at a 95% confidence level. Overall, the present work demonstrated that a flavoursome protein ingredient (i.e. Flavourzyme®-MRP) from low-value meat by-product (i.e. beef bone extract) can be successfully incorporated into extruded meat analogues to form meat alternatives with high aroma and taste quality while maintaining fibrous structure. However, further work needs to be done to improve the textural and sensory properties of sausages made from extruded meat alternatives.
High protein Chinese steamed bread : physico-chemical, microstructural characteristics and gastro-small intestinal starch digestion in vitro : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Manawatū, New Zealand
(Massey University, 2019) Mao, Shiyuan
In Asia, high protein low carbohydrate foods are in high demand because their consumption can provide improved nutritional benefits and help maintaining blood glucose levels close to normal. High protein versions of popular, highly consumed food products (staple foods) such as Chinese steamed bread (CSB) can be very useful to improve the health status of our populations. Thus, the objectives of this study were: to develop high protein Chinese steamed bread (HPCSB) using plant protein, dairy protein combinations. The high protein versions of the steamed breads were then compared with control 100% wheat flour based Chinese steamed bread for physico-chemical, microstructural, textural and in vitro starch digestion characteristics. In order to develop HPCSB, plant proteins (soy protein isolate) and dairy proteins (rennet casein and milk protein concentrate) were blended into wheat flour at two different levels. The addition of proteins has led to a change in colour characteristics (L*, a*, b*) and also resulted in a decreased specific volume of the breads. The textural characteristics measured through textural profile analysis of HPCSB showed an increased hardness and gumminess than control. The microstructure of HPCSB was observed to be more compact and had fewer air cells when observed through Scanning Electronic Microscopy. Furthermore, in vitro starch digestion of HPCSB depicted that the addition of proteins was capable of lowering the starch hydrolysis (%) and estimated glycaemic index (eGI), especially for RC I and RC II at significant levels. Addition of both proteins influenced the microstructure of HPCSBs, which in turn affected the textural and starch digestion properties. High protein Chinese steamed bread with low glycaemic properties can be prepared by critically selecting the protein sources with minimum changes in their physical and textural characteristics.
Functional characterisation of constitutive expresser of pathogenesis-related genes 5 : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Biology at Massey University, Palmerston North, New Zealand
(Massey University, 2017) Faisal, Muhammad
As reported previously, CPR5 negatively regulates the onset of leaf death, hypersensitive response, disease resistance and early leaf senescence. cpr5 plants contain aberrant trichomes and higher levels of ROS, SA and JA. Cell-cycle, JA/ET, ABA and sugar signalling are also affected in cpr5 plants. These results suggest that CPR5 is a master regulator of multiple processes. However, how CPR5 manages to exert pleiotropic effects is still poorly understood. The first objective of the current study was the purification of the CPR5 protein to solve its crystal structure. Extensive in silico analyses were carried out and the results showed that CPR5 is predicted to be a membrane protein with 4 or 5 transmembrane (TM) domains. Additionally, CPR5 contains intrinsically disordered regions (IDRs) at its N-terminus. Proteins containing IDRs and TM domains are often difficult to purify for crystallization studies. Therefore, the undesirable regions of CPR5 such as, IDR and TM domains were deleted and a set of 24 constructs were developed. Despite several efforts, none of the CPR5 recombinant proteins were isolated. In addition to predicting IDR and TM domains, in silico results also predicted three NLS-encoding clusters, casein kinase phosphorylation sites, multiple start codons, coiled-coil domains and glycine motifs. To find out the roles of these putative structural elements on CPR5 functions, firstly a CPR5 cDNA was synthesised and termed as SynCPR5. Subsequently, predicted sites or motifs were mutated in SynCPR5 through sitedirected mutagenesis and a set of 25 mutated CPR5 transgenes (cDNA constructs) were developed. Using a complementation strategy, all the constructs were transformed into cpr5- 2 plants. The results show that the complementation of cpr5-2 plants with SynCPR5, fully restored HR-like lesions, wildtype-like trichomes and leaves on SynCPR5 plants. Further physiological characterization such as, transcript abundance of SynCPR5, PR1, PR5 and PDF1.2, leaf area measurements and ploidy levels showed that CPR5 regulates some of its functions and phenotypes quantitatively as well as qualitatively. When compared with the wildtype, better growth (larger leaves) but enhanced disease susceptibility was found in metCPR5 transgenic lines (in which putative start codons were mutated), indicating that CPR5 regulates a balance between growth and resistance. Functional characterization of NLS mutants (nlsCPR5) showed that NLS-encoding clusters are important for CPR5 proper functions. However, current evidence is insufficient to relate their role in CPR5 localization. Moreover, in silico results show that putative NLS clusters are present in the region of CPR5 which were annotated as intrinsically disordered region (IDR). Similar phenotypes shown by both nlsCPR5 and Del63CPR5 (in which the first 63 amino acids of CPR5 including putative NLS were deleted), indicate that the putative NLS clusters could be part of IDR and may have dual functions. Loss-of-function phenotypes shown by coiled-coil domain mutants (ccdCPR5) reinforce the role of coiled-coil domains in CPR5 homo-dimerization. Moreover, in contrast to previous reports, the downregulation of PDF1.2 in the majority of CPR5 complementation lines proposes CPR5 to be a positive regulator of PDF1.2. Based on the results presented in the current study, putative CPR5 IDRs and coiled-coil domains are proposed to facilitate CPR5 dimerization in order to restrict the entry of deregulated cargos into the nucleus. Moreover, these results uncover a novel role of CPR5 in the regulation of balance between plant growth and resistance. Furthermore, this study, for the first time, reports evidence of the requirement of NLS clusters for CPR5 functions.
Investigation into the relationship between ethylene and sulfur assimilation in Arabidopsis thaliana and onion (Allium cepa L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science (with Honours) in Biochemistry at Massey University
(Massey University, 2006) Sanggang, Fiona Ann
The phytohormone ethylene (C2H4) mediates the adaptive responses of plants to various nutrient deficiencies including iron (Fe)-deficiency, phosphorus (P)-deficiency and potassium (K)-deficiency. However, evidence for the involvement this hormone in the sulfur (S) deficiency response is limited to date. In this study, the effect of C2H4 treatment on the accumulation of the S-assimilation enzymes ATP sulfurylase (ATPS). adenosine-5 -phosphosulfate-reductase (APR), O-acetylserine-(thiol)-lyase (OASTL) and sulfite reductase (SiR) was examined in A. thaliana and onion (A. cepa). To complement this, the effect of short-term S-depletion on the expression of the 12-member gene family of the C2H4 biosynthetic enzyme, l-amino-cyclopropane-l-carboxylic acid (ACC) synthase (ACS) from A. thaliana, designated AtACS1-12, was also examined. Western analyses were used to show that plants of A. thaliana pre-treated with the C2H4-signalling inhibitor 1-MCP, had elevated levels of ATPS, APR and OASTL protein in leaf tissue at all time points examined, suggesting that C2H4 has an inhibitory effect on the accumulation of these enzymes. However, SiR appeared to be under dual regulation by C2H4: under S-sufficient conditions C2H4 appears to prevent the unnecessary accumulation of SiR and conversely promote the fast accumulation of SiR under S-depleted conditions. The changes in AtACS1-12 expression in the root and leaf tissues of S-sufficient and S-depleted plants of A. thaliana were examined by RT-PCR using gene-specific, exon-spanning primers. The expression patterns of AtACS2, AtACS6 and AtACS7 were comparable regardless of S availability and may therefore be housekeeping genes. In contrast, the expression of AtACS5 in leaf, and AtACS8 and AtACS9 in roots was repressed under S-depleted conditions, although the mechanism of this repression cannot be elucidated from this study. The protein products of these closely-related genes are believed to be phosphorylated and stabilised by a CDPK whose activity may be compromised by S-depletion. The inhibition of AtACS5, AtACS8 and AtACS9 expression, and the decrease in AtACS5, AtACS8 and AtACS9 accumulation, and hence less C2H4 production, may be part of the plant adaptive response to S-depletion, as the C2H4 -mediated repression of root growth is alleviated to allow the plant to better seek out the lacking nutrient. The expression of the MPK-stabilised genes AtACS2 and AtACS6 appeared to be similar regardless of S availability, although this may merely be a consequence of the scoring method used in this study, which cannot determine whether there was any difference in the level of expression of these genes. The expression of AtACS10 and AtACS12 was repressed in S-deficient plants. Although both AtACS10 and AtACS12 isozymes posses the hallmark seven conserved regions found in the ACSes of other plant species, they are also phylogenetically related to alanine and aspartate aminotransferases, and are known to encode aspartate (AtACS10) and aromatic amino acid transaminases (AtACS12). Therefore, the apparent downregulation of these genes suggests that the downregulation of amino acid metabolism may be part of the plant adaptive response to S-depletion. The downregulation of several AtACS genes, and therefore possibly also C2H4 biosynthesis, in S-deficient plants was accompanied by an accumulation of APR protein. The increase in APR protein that also occurred in 1-MCP-treated plants indicates that C2H4 may be involved in the plant response to S-depletion, because in both cases the upregulation of the S-assimilation pathway, as manifested by the accumulation of APR protein, occurred when C2H4 biosynthesis and signalling was repressed. However, the possible role of other phytohormoes in the plant response to S-depletion cannot be excluded, as there is evidence for crosstalk between the C2H4 signalling pathway and those of auxin, abscisic acid (ABA), cytokinins and jasmonic acid (JA). Furthermore, because C2H4 has been implicated in the response of various plants to Fe-deficiency, P-deficiency, and K-deficiency, in addition to S-deficiency, it may be a regulator of the plant adaptive response to nutrient stresses in general.
Studies on abscission cell differentiation in Sambucus nigra and Phaseolus vulgaris : submitted in partial fulfilment of the degree of Doctor of Philosophy in Plant Biology, Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand
(Massey University, 2002) Flight, Simone
This thesis examines aspects of abscission cell differentiation in Sambucus nigra and Phaseolus vulgaris. The experimentation is divided into two sections; an in vivo study examining the cell wall proteins from the leaf rachis abscission zones of S. nigra, to identify proteins that denote the abscission zone as a fully differentiated cell type, and an in vitro study examining aspects of secondary or adventitious abscission zone formation in petiole explants of P. vulgaris. As an initial approach to identify abscission cell-specific proteins, a survey of the total cell wall bound proteins in four tissues, leaf mid-rachis (MR), ethylene-treated leaf mid-rachis (MRE), 0 h, or freshly excised leaf rachis abscission zone (OZ) and ethylene-treated abscission zone (ZONE) was undertaken. The study also involved surveying these tissues over the vegetative seasons (spring, summer, autumn). Separation of these protein extracts using SDS-PAGE revealed proteins that were putatively uniquely expressed in each of the tissues. Moreover, the expression of some proteins changed from spring through to autumn. Further fractionation of the extracts using hydrophobic interaction chromatography (HIC), and separation of the fractions using SDS-PAGE, illustrated there were many more proteins that had not been resolved in the initial survey of wall extracts. In total, four proteins of ca. 10, 28, 38 and 43 kDa were identified in the UZ tissue only and six proteins (ca. 10, 34, 36, 40, 74 and 75 kDa) were detectable in the OZ and ZONE tissues. Three of the putative OZ-specific proteins (designated OZ10, OZ28 and OZ43) were trypsin-digested and some initial amino acid sequence data obtained. The OZ10 tryptic fragment had closest identity to a lipid transfer protein (LTP) from spinach, and the OZ43 fragment had closest identity to an aldose-1-epimerase-like protein expressed in tobacco. Two peptides were sequenced from the OZ28 protein; one had highest identity to a superoxide dismutase and the second had identity to a ribonuclease. Two of these, OZ10 and OZ43 were characterised further. Antibodies raised to LTPs protein from Daucus carota and Arabidopsis thaliana recognized a protein of 10 kDa that was expressed in both the rachis and abscission zone tissues of S. nigra before and after ethylene treatment. Moreover, the LTP antibodies detected a ca. 10 kDa protein in freshly excised and ethylene-treated distal pulvinus, primary abscission zone and petiole tissues of P. vulgaris with highest expression in ethylene-treated petiole tissue. The second protein, to be characterised further was most similar in sequence to a nuclear pore membrane protein identified in tobacco suspension cells and designated gp40. This protein appears to be an aldose-1-epimerase-like enzyme (otherwise known as mutarotase) from its homology to bacterial forms of mutarotase. An antibody to gp40 recognized a ca. 43kDa protein in the non-ethylene treated rachis and zone cell wall extracts of S. nigra, the putative OZ43. This same antibody did not recognize any proteins in the protein extract from porcine and lamb kidney, tissues that have mutarotase activity. A coupled enzyme assay was developed to measure the mutarotase activity in the plant samples. Although mutarotase activity was measured in both the soluble and cell wall bound fractions of the rachis cells, purification of the OZ43 protein using column chromatography or through cell fractionation revealed that the ca. 43 kDa protein recognised by the gp40 antibody did not appear to be responsible for this activity. For the second part of this thesis, the in vitro study, the aim was to measure the levels of IAA, ethylene and ACC oxidase enzyme activity in bean petioles explants during IAA-induced secondary abscission zone formation. In the bean explant system, the secondary zone forms at a site along the petiole which is removed from the primary zone and governed by the concentration of IAA added. The petiole tissue that links the primary zone with the secondary zone (the distal segment) remains green and, in this thesis, is designated as G1. The petiole tissue proximal to the zone senesces and yellows, and is divided into Y2 (immediately proximal to the secondary zone), Y3 (mid way) and Y4 (the most proximal petiole tissue). To measure changes in IAA concentration during secondary zone formation, an immunoassay (ELISA) was developed, initially using polyclonal antibodies to IAA, but the titre of these antibodies was not sufficient and so monoclonal antibodies were used. During secondary zone formation, the concentration of free IAA in the petiole tissue changed dramatically, with measurements ranging between 6 and 2608 pmol/g fresh weight (FW) of tissue. The IAA concentration in the petioles at separation at the primary zone before IAA was added was lower in the G1 and Y2 sections (ca. 30 pmol/g FW) when compared with the Y3 and Y4 sections (166 and 271 pmol/g FW respectively). At 6 h after the application of IAA, the concentration of IAA had increased to 146 pmol/g FW in the G1 section, remained the same in the Y2 section and increased to 208 and 423 pmol/g FW in the Y3 and Y4 sections, respectively. At 26 h after the application of IAA, and approximately the time of initiation of differentiation of the secondary zone, the IAA concentration was similar to the petioles after 6 h (179 and 21 pmol/g FW for G1 and Y2 respectively) and significantly lower in the Y3 and Y4 sections (35 and 69 pmol/g FW respectively). At the first point at which the green:yellow tissue can be ascertained (at 52 h) the IAA concentration was dramatically higher in the G1 and Y2 tissues (1125 and 1090 pmol/g FW respectively) compared to measurements in the Y3 and Y4 sections at 52 h of 17 and 107 pmol/g FW respectively. At separation at the secondary abscission zone, the IAA measurements in the G1, Y2 and Y4 sections were 405, 315 and 1198 pmol/g FW respectively. The ethylene produced from freshly excised pulvinus and petiole tissue was ca. 0.20 nmol/h/g FW and increased to 1.7 nmol/h/g FW in the pulvinus, 0.39 nmol/h/g FW in the G1 petiole section and 0.67 nmol/h/g FW in the Y2/Y3/Y4 pooled petiole sections at separation at the primary zone. At separation of the secondary zone, ethylene evolution measurements of 1.73 nmol/h/g FW in G1 and 4.37 nmol/h/g FW in the Y2/Y3/Y4 tissue were observed. However, the activity and expression of ACC oxidase was higher in the fresh tissues and non-senescent petiole region (G1), but was lowest in the senescent (Y2,Y3 and Y4) tissue at the formation of the secondary zone.
An evaluation of lupins (Lupinus spp.) for seed protein production : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Massey University, Palmerston North, New Zealand
(Massey University, 1979) Withers, Neville John
Since 1972 there has been interest in the greater use of seed protein in grain-based meals for stock. Lupins were one of the crops proposed to fill this requirement. This study was initiated to provide information on the agronomic requirements of Lupinus angustifolius, L. luteus and L. albus for seed production with emphasis on the southern North Island of New Zealand. In addition, some more basic studies on carbon and nitrogen translocation and the response of lupins to water stress were also carried out to provide a better understanding of the lupin plant and its response to its environment. Initially some field experiments were laid down to measure responses to sowing date, plant density, defoliation and cultivar. At wide spacing, L. angustifolius showed an approximately linear decrease in seed yield/plant as sowing date moved from April to October. At normal densities, however, sowing in late July gave the best see yield. Autumn sowings were affected by disease. It was concluded that, in the absence of disease, seed yield was largely determined by the length of the period of favourable environmental conditions between the start of flowering and the finish of reproductive development. This period determined the number of lateral inflorescences produced which, in turn, determined the number of pods producing seed. Pod number was the main component influencing seed yield. Thus, early sowing and reliable summer rainfall or irrigation seem to be the factors determining high lupin seed yields. Responses to density were variable. In one experiment there was no response in seed yield by four cultivars over these sowing times to densities ranging from 50-140 pl/m2. In a further experiment, increases in seed yield were obtained as plant density increased from 25-100 pl/m2. Removal of the main stem growing point early in growth briefly stimulated lateral stem growth but the effect on lateral stem seed yield was insufficient to compensate for the loss of the main stem seeds. There was little difference between the L. angustifolius cultivars Uniharvest, Uniwhite and Unicrop when sown early but, with late spring sowing, Unicrop flowered earlier which was an advantage under dry early summer conditions. In one experiment comparing a range of legume species, L. albus and Pisum sativum produced the highest seed yield but L. albus and L. luteus yielded the most protein per unit area. The peak rate of nitrogen accumulation in all species was similar and the main factor influencing protein yield appeared to be the duration of nitrogen accumulation. Provided each crop utilised similar durations of the growing period, the yield of seed protein/ha from various legume crops is likely to be similar; the main difference being the composition of the seed. It was suggested that, for maximum seed protein yield, indeterminate cultlvars may have some advantage over more determinate cultivars provided appropriate management procedures are adopted. Studies on water stress indicated that it plays an important role by influencing the distribution of assimilate between vegetative and reproductive growth. Mild water stress tended to stop vegetative growth and increase the rate of seed growth. When sufficiently severe, water stress appeared to initiate the senescence of the plant, the timing of which determined the potential seed yield for that situation. Water deficit had its main effect on seed yield by reducing pod number. Other yield components were relatively stable. Day temperatures of 28°C, when imposed early in growth, reduced vegetative and seed yield in L. albus. As the plant developed, however, the adverse effects of high temperature decreased until growth was stimulated during first order lateral flowering. No direct effect of high temperature on pod abscission was apparent and it was suggested that pod loss under high temperatures which have been reported occurred largely because of an associated water stress. A 14C translocation study indicated that most movement of photosynthate in L. albus was into the branch on which the labelled leaf was inserted, or into lower branch orders directly connected to it. Results suggest that, in L. albus cv. Ultra, lower order stems are a more important competitor with the inflorescence for photosynthate than the new, rapidly developing, higher order lateral branches. A possible strategy for growing lupin in a commercially viable situation in the Southern North Island is discussed.
Molecular genetic analysis of the maize terminal ear1 gene and in silico analysis of related genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor in Philosophy in Plant Biology at Massey University, Palmerston North, New Zealand
(Massey University, 2001) Jeffares, Daniel Charlton
Mutants of the maize terminal ear1 (te1) gene have shortened internodes, abnormal phyllotaxy, leaf pattern defects and partial feminisation of tassels. The te1 gene encodes an RNA recognition motif (RRM) protein, and is expressed in the vegetative shoot apex in semicircular rings that laterally oppose the positions of leaf primordia (Veit 1998). This project aimed to further characterise the molecular biology and function of the te1 gene. Molecular genetic studies aimed to further characterise the genes structure and expression. Genomic clones were sequenced revealing the intron exon structure. 5' RACE was used to predict a 5' transcription start site. Competitive RT-PCR showed that te1 transcripts were highest in vegetative shoot meristems and embryos, lower in ears, roots and tassels, and undetectable in leaves. Two te1 mutant alleles were cloned and the junctions sequenced, a further five alleles were characterised incompletely. The TE1 peptide belongs to a subclass of RRM proteins which includes the Schizosaccharomyces pombe protein MEI2. More than 30 putative plant Mei2-like genes were identified in Genbank, no examples have been found in metazoans. Seven Mei2-like genes were predicted from the completed Arabidopsis genome. Exon structure and amino acid sequence supported three groupings of Mei2-like genes. Structural predictions of Mei2-like proteins indicate that the third RRM contained some novel structural features not present in canonical RRM proteins. Attempts to study the function of the TE1 protein in vitro were limited by the inability of both E. coli and Pichia pastoris expression systems to express the full length protein, probably due to codon bias. Antibodies produced to a C-terminal portion of the protein did not specifically detect the TE1 protein in plant extracts without incurring non-specific activity. The te1 cDNA was ectopically expressed in Arabidopsis from a copper-inducible promoter both with and without the SV40 nuclear localisation signal (NLS). Although both te1 and NLS:te1 transgenes were detected in transformants no phenotypes consistently correlated with transgene expression.

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