Responses to phosphate deprivation in white clover (Trifolium repens L.) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Plant Biology at Massey University

Four breeding lines of white clover (Trifolium repens L.) were obtained from AgResearch Grasslands, Palmerston North, New Zealand, that had been shown previously to differ in terms of specific growth responses to added phosphate (P) in the field. These were designated Breeding Line (BL) 43 (low performer on low P; low performer on high P), BL 45 (low performer on low P; high performer on high P), BL 47 (high performer on low P; high performer on high P), and BL 49 (high performer on low P; low performer on high P). These breeding lines and five selected genotypes that were propagated from each line (designated 43-7, 43-8. 45-14. 45-4 and 47-9) were rooted in half-strength Hoagland solution in vermiculite for two weeks and then transferred to half-strength Hoagland liquid media for five weeks prior to the initiation of the experiments. For the breeding line screening, plants were acclimatized in a constant temperature environment for one week prior to treatments, while for the genotypic screening, plants were maintained in a temperature-controlled glasshouse. These lines and genotypes were characterized in relation to P uptake and utilization efficiency by growing in P-sufficient media (+P; 0.5 mM KH2PO4) and P-deficient media (-P; 0 mM KH2PO4) for 3, 5, 7 and 14 days (for the breeding line screening) and 7, 14 and 21 days (for the genotype screening). Over the time course, inorganic phosphate (Pi) content in leaves, non-specific acid phosphatase (APase) activity in intact roots (both as a total soluble activity and a cell-wall-associated activity), isoenzyme analyses, shoot dry weight (DW) and fresh weight (FW), leaf area, weight of an individual leaf (designated as the weight of the first fully expanded leaf), root FW, and the root:shoot (R:S) ratio were determined. Pi deprivation enhanced the induction of one major low mobility cell wall acidic isoform, two minor high mobility cell wall acidic isoforms and one major low mobility cell wall basic isoform in all genotypes. Furthermore, the activity of one major low mobility cell wall basic isoform was more higher in genotype 45-14 and one minor high mobility cell wall basic isoform was induced only in genotype 45-14 in response to Pi deprivation. In terms of individual BLs and genotypes, the screening results showed that BL 49 and genotype 45-14 displayed a constant Pi content and a slow induction of APase activity in the -P media, and had the highest total biomass FW in both +P and -P media. Overall (in both treatments) BL 49 and genotype 45-14 are the most efficient at utilizing available P as they produced the largest biomass FW, produced more roots in P- deprived media when compared with the other BLs and genotypes, and were more efficient in utilizing the P for the synthesis of biomass. BLs 43 and 45 and genotypes 43-7 and 43-8 are less efficient at utilizing available P, while under P deprivation, BL 45 and genotype 45-14 are the most efficient at utilizing P compared to the other BLs and genotypes. The study also showed that the Pi content in leaves and APase activity in roots was found to be the plant parameter most sensitive to Pi deprivation, and the results suggest that the selection of white clover germplasm for satisfactory performance under low P availability can be carried out using these two parameters as criteria

Functional Classification of Skinning Injury Responsive Genes in Storage Roots of Sweetpotato

Skinning injury in sweetpotato due to loss of skin or periderm which occurred during harvest is inevitable and account for financial loss due to dehydration, pests, and pathogens. Hence, studies on gene expression changed due to skinning injury can provide important information about this protective tissue and for improving the life of storage roots. New candidate genes involved in skinning injury were isolated with an Annealing Control Primer (ACP). Using 20 ACP primers, a total of 103 differentially expressed genes (DEGs) were retrieved. In this study, the functional annotation of these selected 15 up-regulated DEGs (10 contigs and 5 singletons) were characterized. The results showed that these 15 “DEG-unigenes” are mainly associated with defense and stress responses, regulation and signaling, protein synthesis and fate, and metabolism may play an important role in the primary responses to skinning injury in storage roots of sweetpotato. This study showed the importance of defense and stress responses genes to the formation of wound periderm. Furthermore, this results can be used for better understanding of the molecular mechanism of skinning/mechanical injury-related genes in the storage roots of sweetpotato as well as to all stems, fruits, and roots of all plants. Keywords: differentially expressed gene, gene function, Ipomoea batatas, woundin

Skinning Injury Responses in Sweetpotato

In sweetpotatoes (Ipomoea batatas L. Lamb), the loss of skin from the surface of the storage roots is known as skinning injury. It is responsible for significant postharvest loss resulting from moisture increase and weight reduction, wrinkling, and susceptibility to pathogen attack. Reduced root weight by water loss is associated with a higher rate of rot predominantly occurred in the developing and underdeveloped countries which can count of 8-20% of postharvest loss. Plants have different adaptation to protect themselves against skinning injury. Lignification, suberization, and increased sugar at the wound site have been shown to be correlated with wound healing. Changing in gene expressions have been associated with skinning injury. Genes associated in the biosynthesis of lignin and suberin, protein fate, cell-wall modification, transcription and protein synthesis, and stress responses and defense have been associated with skinning injury responses in plants. Understanding the skinning injury responses and how to regulate them can be used to produce a more desirable plant resistant to skinning injury. This paper especially reviews and discusses skinning injury responses in sweetpotato, a root crop which product may severely be affected by skinning injury. Keywords: gene expression, Ipomoea batatas, lignification, postharvest loss, wounding   ABSTRAK Pada ubi jalar (Ipomoea batatas L. Lamb), cedera kulit adalah hilangnya kulit dari permukaan umbi. Cedera kulit ini bertanggung jawab atas kerugian pascapanen yang signifikan akibat peningkatan laju kelembaban dan penurunan berat umbi, pengerutan, dan kerentanan terhadap serangan patogen. Berat umbi yang berkurang karena kehilangan air dikaitkan dengan tingkat pembusukan yang lebih tinggi, terutama terjadi di negara-negara berkembang dan yang kurang berkembang dengan kehilangan hasil panen umbi 8-20%. Tanaman memiliki adaptasi yang berbeda untuk melindungi diri dari cedera kulit. Lignifikasi, suberisasi, dan peningkatan gula di lokasi pelukaan telah terbukti berkorelasi dengan penyembuhan luka. Perubahan ekspresi gen telah dikaitkan dengan cedera kulit. Gen-gen yang terlibat dalam jalur biosintesis lignin dan suberin, protein tujuan akhir, modifikasi dinding sel, transkripsi dan sintesis protein, serta respons stres dan pertahanan telah dikaitkan dengan respons cedera kulit pada tanaman. Memahami respons cedera kulit dan bagimana cara mengaturnya dapat digunakan untuk menghasilkan tanaman yang diinginkan yang tahan terhadap cedera kulit umbi. Paper ini secara khusus mengulas dan membahas respon cedera kulit pada ubi jalar, suatu tanaman umbian yang hasilnya sangat terpengaruh oleh cedera kulit. Kata kunci: ekspresi gen, Ipomoea batatas, lignifikasi, kehilangan pascapanen, pelukaa

Functional Classification of Skinning Injury Responsive Genes in Storage Roots of Sweetpotato

Skinning injury in sweetpotato due to loss of skin or periderm which occurred during harvest is inevitable and account for financial loss due to dehydration, pests, and pathogens. Hence, studies on gene expression changed due to skinning injury can provide important information about this protective tissue and for improving the life of storage roots. New candidate genes involved in skinning injury were isolated with an Annealing Control Primer (ACP). Using 20 ACP primers, a total of 103 differentially expressed genes (DEGs) were retrieved. In this study, the functional annotation of these selected 15 up-regulated DEGs (10 contigs and 5 singletons) were characterized. The results showed that these 15 “DEG-unigenes” are mainly associated with defense and stress responses, regulation and signaling, protein synthesis and fate, and metabolism may play an important role in the primary responses to skinning injury in storage roots of sweetpotato. This study showed the importance of defense and stress responses genes to the formation of wound periderm. Furthermore, this results can be used for better understanding of the molecular mechanism of skinning/mechanical injury-related genes in the storage roots of sweetpotato as well as to all stems, fruits, and roots of all plants