Yuchan Zhou

Review: Nutrient loading of developing seeds

Interest in nutrient loading of seeds is fuelled by its central importance to plant reproductive success and human nutrition. Rates of nutrient loading, imported through the phloem, are regulated by transport and transfer processes located in sources (leaves, stems, reproductive structures), phloem pathway and seed sinks. During the early phases of seed development, most control is likely to be imposed by a low conductive pathway of differentiating phloem cells serving developing seeds. Following the onset of storage product accumulation by seeds, and, depending on nutrient species, dominance of path control gives way to regulation by processes located in sources (nitrogen, sulfur, minor minerals), phloem path (transition elements) or seed sinks (sugars and major mineral elements, such as potassium). Nutrients and accompanying water are imported into maternal seed tissues and unloaded from the conducting sieve elements into an extensive post-phloem symplasmic domain. Nutrients are released from this symplasmic domain into the seed apoplasm by poorly understood membrane transport mechanisms. As seed development progresses, increasing volumes of imported phloem water are recycled back to the parent plant by process(es) yet to be discovered. However, aquaporins concentrated in vascular and surrounding parenchyma cells of legume seed coats could provide a gated pathway of water movement in these tissues. Filial cells, abutting the maternal tissues, take up nutrients from the seed apoplasm by membrane proteins that include sucrose and amino acid/H+ symporters functioning in parallel with non-selective cation channels. Filial demand for nutrients, that comprise the major osmotic species, is integrated with their release and phloem import by a turgor-homeostat mechanism located in maternal seed tissues. It is speculated that turgors of maternal unloading cells are sensed by the cytoskeleton and transduced by calcium signalling cascades.

Enzymes associated with blackheart development in pineapple fruit

The involvement of browning enzymes, polyphenoloxidase (PPO), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in blackheart development was investigated in pineapple fruit (Ananas comosus, Smooth Cayenne) following low temperature storage. An increase in PPO activity was related to the incidence of blackheart symptoms, both temporally and spatially. Fruit maturity significantly affected blackheart susceptibility; immature and over-mature fruits developed less blackheart injury than in ature fruit. The effect of maturity on blackheart susceptibility was highly correlated to the response of PPO activity to chilling. POD showed no significant change after chilling. Enhanced PAL activity was observed during chilling at 6, 13 and 18 degreesC. Chilling (6, 13 and 18 degreesC) also inhibited the increase of ascorbate peroxidase activity observed in the fruit stored continuously at 25 degreesC, but had less effect on catalase activity. The results indicate that the development of blackheart symptoms in pineapple fruit results from the disturbance of a number of metabolic processes that occur at sub-ambient temperatures

Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons

Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H+ symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.

An epidermal-specific ethylene signal cascade regulates trans-differentiation of transfer cells in Vicia faba cotyledons

*Transfer cells (TCs) trans-differentiate by developing extensive wall ingrowths that facilitate enhanced plasma membrane transport of nutrients. Signal(s) and signalling cascades responsible for initiating this trans-differentiation event are poorly understood. We tested the hypothesis that ethylene functions as a key inductive signal for wall ingrowth formation in epidermal cells of Vicia faba cotyledons. *Scanning electron microscopy of epidermal cells monitored their propensity for wall ingrowth formation. Spatial and temporal expression profiles of ethylene biosynthetic enzymes and key elements of ethylene signalling cascades (ethylene insensitive 3 (EIN3) and ethylene response factors (ERFs)) were determined. *Wall-ingrowth formation responded positively to manipulation of ethylene biosynthesis and perception. It was preceded by a cell-specific burst in ethylene biosynthesis accompanied by a co-localized post-translational up-regulation of VfEIN3-1 and differential expression of three VfERF genes. Blocking ethylene production arrested ongoing wall ingrowth development. Wound-induced ethylene in pod walls and seed coats caused an in planta activation of ethylene biosynthetic genes in adaxial epidermal cells that coincidentally formed wall ingrowths. *A cell-specific burst of ethylene biosynthesis functions as an inductive signal initiating and sustaining trans-differentiation to a TC morphology in vitro. These events are reproduced for developing V. faba seeds in planta.

Early gene expression programs accompanying trans‐differentiation of epidermal cells of Vicia faba cotyledons into transfer cells

Transfer cells (TCs) trans-differentiate from differentiated cells by developing extensive wall ingrowths that enhance plasma membrane transport of nutrients. Here, we investigated transcriptional changes accompanying induction of TC development in adaxial epidermal cells of cultured Vicia faba cotyledons. Global changes in gene expression revealed by cDNA-AFLP were compared between adaxial epidermal cells during induction (3 h) and subsequent building (24 h) of wall ingrowths, and in cells of adjoining storage parenchyma tissue, which do not form wall ingrowths. A total of 5795 transcript-derived fragments (TDFs) were detected; of these, 264 TDFs showed epidermal-specific changes in gene expression and a further 207 TDFs were differentially expressed in both epidermal and storage parenchyma cells. Genes involved in signalling (auxin/ethylene), metabolism (mitochondrial; storage product hydrolysis), cell division, vesicle trafficking and cell wall biosynthesis were specifically induced in epidermal TCs. Blockers of auxin action and vesicle trafficking inhibited ingrowth formation and marked increases in cell division accompanied TC development. Auxin and possibly ethylene signalling cascades induce epidermal cells of V. faba cotyledons to trans-differentiate into TCs. Trans-differentiation is initiated by rapid de-differentiation to a mitotic state accompanied by mitochondrial biogenesis driving storage product hydrolysis to fuel wall ingrowth formation orchestrated by a modified vesicle trafficking mechanism.

Dual gene expression cassette vectors with antibiotic selection markers for engineering in Saccharomyces cerevisiae

BackgroundManipulations in Saccharomyces cerevisiae classically depend on use of auxotrophy selection markers. There are several disadvantages to this in a microbial cell factory setting: (1) auxotrophies must first be engineered in prototrophic strains, and many industrial strains are polyploid/aneuploid prototrophs (2) available strain auxotrophies must be paired with available repair plasmids (3) remaining auxotrophies must be repaired prior to development of industrial bioprocesses. Use of dominant antibiotic resistance markers can circumvent these problems. However, there are relatively few yeast antibiotic resistance marker vectors available; furthermore, available vectors contain only one expression cassette, and it is often desirable to introduce more than one gene at a time.ResultsTo overcome these problems, eight new shuttle vectors have been developed. The plasmids are maintained in yeast under a 2 μm ori and in E. coli by a pUC ori. They contain two yeast expression cassettes driven by either (1) the constitutive TEF1 and PGK1 promoters, or (2) the constitutive TEF1 promoter and the inducible GAL10 or HXT7 promoters. Expression strength of these promoters over a typical production time frame in glucose/galactose medium was examined, and identified the TEF1 and HXT7 promoters as preferred promoters over long term fermentations. Selection is provided by either aphA1 (conferring resistance to G418 in yeast and kanamycin/neomycin in E. coli) or ble (conferring resistance to phleomycin in both yeast and E. coli). Selection conditions for these plasmids/antibiotics in defined media were examined, and selection considerations are reviewed. In particular, medium pH has a strong effect on both G418 and phleomycin selection.ConclusionsThese vectors allow manipulations in prototrophic yeast strains with expression of two gene cassettes per plasmid, and will be particularly useful for metabolic engineering applications. The vector set expands the (currently limited) selection of antibiotic marker plasmids available for use in yeast, and in addition makes available dual gene expression cassettes on individual plasmids using antibiotic selection. The resistance gene cassettes are flanked by loxP recognition sites to allow CreA-mediated marker removal and recycling, providing the potential for genomic integration of multiple genes. Guidelines for selection using G418 and phleomycin are provided.

Breadfruit (Artocarpus altilis) gibberellin 2-oxidase genes in stem elongation and abiotic stress response

Breadfruit (Artocarpus altilis) is a traditional staple tree crop in the Oceania. Susceptibility to windstorm damage is a primary constraint on breadfruit cultivation. Significant tree loss due to intense tropical windstorm in the past decades has driven a widespread interest in developing breadfruit with dwarf stature. Gibberellin (GA) is one of the most important determinants of plant height. GA 2-oxidase is a key enzyme regulating the flux of GA through deactivating biologically active GAs in plants. As a first step toward understanding the molecular mechanism of growth regulation in the species, we isolated a cohort of four full-length GA2-oxidase cDNAs, AaGA2ox1- AaGA2ox4 from breadfruit. Sequence analysis indicated the deduced proteins encoded by these AaGA2oxs clustered together under the C19 GA2ox group. Transcripts of AaGA2ox1, AaGA2ox2 and AaGA2ox3 were detected in all plant organs, but exhibited highest level in source leaves and stems. In contrast, transcript of AaGA2ox4 was predominantly expressed in roots and flowers, and displayed very low expression in leaves and stems. AaGA2ox1, AaGA2ox2 and AaGA2ox3, but not AaGA2ox4 were subjected to GA feedback regulation where application of exogenous GA3 or gibberellin biosynthesis inhibitor, paclobutrazol was shown to manipulate the first internode elongation of breadfruit. Treatments of drought or high salinity increased the expression of AaGA2ox1, AaGA2ox2 and AaGA2ox4. But AaGA2ox3 was down-regulated under salt stress. The function of AaGA2oxs is discussed with particular reference to their role in stem elongation and involvement in abiotic stress response in breadfruit.

Dwarfing of Breadfruit (Artocarpus altilis) Trees: Opportunities and Challenges

Breadfruit [ Artocarpus altilis (Parkinson) Fosberg)] is a traditional staple crop grown for its starchy fruit throughout the tropics. It has long been recognized for its potential to alleviate hunger in the region. However, being a tree of 10 ‐ 30m, breadfruit is vulnerable to wind damage. Owing to the continuing trend of global climate change, the success of the species as a sustainable crop for delivering local food security is compromised by the likelihood of more intense tropical windstorms in the island nations. Tree height also forms a major constraint to disease management and fruit harvesting. These imperatives have driven an increasing interest in developing breadfruit varieties with short stature. While a great diversity of breadfruit cultivars with varying nutritional and agronomic characteristics exists, the genetic resource showing dwarfing traits is largely uncharacterised. Historically, there has been no intentional breeding for breadfruit cultivars. The long growth cycle, predominantly vegetative propagation and lack of genome information create challenge for crop improvement through traditional breeding. In this review, we highlight the current knowledge of plant dwarfism and its application in agricultural practices and genetic improvement for dwarf phenotype, and present options and tools for breadfruit dwarfing with special reference to natural genetic variability for dwarfing rootstocks, plant growth regulators, potential of mutagenesis and its combination with the currently

Breadfruit (Artocarpus altilis) gibberellin 20-oxidase genes: sequence variants, stem elongation and abiotic stress response

Breadfruit (Artocarpus altilis) is a traditional staple tree crop throughout the tropics. Susceptibility to windstorm damage is the primary constraint on breadfruit cultivation. Significant tree loss due to intense tropical windstorm in the past decades has driven an increasing interest in developing dwarf varieties of breadfruit. As a first step toward understanding the molecular mechanism of growth regulation in the species, we investigated the role of gibberellin and the regulation of GA20-oxidase genes in breadfruit. We provided first evidence that the stem elongation in breadfruit could be manipulated by exogenous gibberellin-related growth regulators. We then cloned six GA20-oxidase cDNAs, AaGA20ox1–AaGA20ox6, in full length from breadfruit. Sequence analysis showed that the predicted proteins of the AaGA20ox1–AaGA20ox3 bear all the hallmarks of functional GA20-oxidase of other species, but predicted AaGA20ox4–AaGA20ox6 as expressed, unprocessed pseudogenes closely related to AaGA20ox2. AaGA20ox1, AaGA20ox3 and AaGA20ox4 were predominantly expressed in green vegetative organs, but displayed different expression pattern in roots and reproductive organs. AaGA20ox2, AaGA20ox5 and AaGA20ox6 were expressed mainly in leaves at low level. AaGA20ox1, AaGA20ox3–AaGA20ox6 were subjected to GA feedback regulation following treatment of exogenous gibberellin and/or gibberellin biosynthesis inhibitors. AaGA20ox1 and AaGA20ox3 were down-regulated under drought and salinity stress, but AaGA20ox2 was up-regulated under salt stress. Pseudogenes AaGA20ox4 and AaGA20ox5 were up-regulated under drought or/and salt stress condition. The function of AaGA20oxs is discussed with particular reference to their role in stem elongation and involvement in abiotic stress response in breadfruit.

Breadfruit (Artocarpus altilis) DELLA genes: gibberellin-regulated stem elongation and response to high salinity and drought

Breadfruit (Artocarpus altilis) is a traditional staple tree crop in the Oceania. Susceptibility to tropical windstorm damage has driven an interest in developing breadfruit with dwarf stature. Gibberellin (GA) is one of the most important determinants of plant height and DELLA proteins are repressors of GA signalling pathway. As a first step toward understanding the molecular mechanism of growth regulation in the species, we isolated two full-length DELLA cDNAs, AaDELLA1 and AaDELLA2 from breadfruit. Sequence analysis indicated the deduced proteins encoded by these AaDELLAs bear all the hallmarks of functional DELLA of other species. Transcripts of AaDELLA1 and AaDELLA2 were detected in all plant organs, but the expression was much lower in flowers and fruits. AaDELLA1 showed predominantly higher expression in roots, but AaDELLA2 displayed higher expression in stems and leaves. Expression of both AaDELLAs was not significantly changed by exogenous GA3 treatment. Under paclobutrazol treatment, the expression of AaDELLA2 was up-regulated, but the expression of AaDELLA1 was not changed significantly. Treatments of high salinity up-regulated the expression levels of both AaDELLA1 and AaDELLA2. Treatment of drought stress increased the expression of AaDELLA2, but not that of AaDELLA1. The function of AaDELLAs is discussed with particular reference to their role in stem elongation and the opportunities of breadfruit dwarfing.