Evelyn Mae Tecson-Mendoza

The protein kinase Pstol1 from traditional rice confers tolerance of phosphorus deficiency

As an essential macroelement for all living cells, phosphorus is indispensable in agricultural production systems. Natural phosphorus reserves are limited, and it is therefore important to develop phosphorus-efficient crops. A major quantitative trait locus for phosphorus-deficiency tolerance, Pup1, was identified in the traditional aus-type rice variety Kasalath about a decade ago. However, its functional mechanism remained elusive until the locus was sequenced, showing the presence of a Pup1-specific protein kinase gene, which we have named phosphorus-starvation tolerance 1 (PSTOL1). This gene is absent from the rice reference genome and other phosphorus-starvation-intolerant modern varieties. Here we show that overexpression of PSTOL1 in such varieties significantly enhances grain yield in phosphorus-deficient soil. Further analyses show that PSTOL1 acts as an enhancer of early root growth, thereby enabling plants to acquire more phosphorus and other nutrients. The absence of PSTOL1 and other genes—for example, the submergence-tolerance gene SUB1A—from modern rice varieties underlines the importance of conserving and exploring traditional germplasm. Introgression of this quantitative trait locus into locally adapted rice varieties in Asia and Africa is expected to considerably enhance productivity under low phosphorus conditions.

Extraction of genomic DNA from the lipid-, polysaccharide-, and polyphenol-rich coconut (Cocos nucifera L.)

One prerequisite to reliable molecular biology work is that the genomic DNA of a sample be of good quality. Coconut is quite difficult to work on because of the high lipid and polysaccharide content of its endosperm and the high polyphenol content of its leaves. This study aimed to determine which protocol to use and which part of the coconut tree is most appropriate to extract good-quality genomic DNA. Genomic DNA from the solid endosperm was found to be of poor quality because of high levels of lipid and galactomannan contaminants. By using a modified protocol by Cheung et al. (1993) as modified by Rogers et al. (1996) and by Dellaporta et al. (1983) as modified by Datta et al. (1997), genomic DNA extracted from the young leaves of the first emergent frond provided enzyme-digestible, good-quality DNA. The modification involved the use of a higher salt concentration (2 M instead of 0.5 M) in the extraction buffer and the use of polyvinylpolypyrrolidone. Moreover, this modified protocol did not involve the use of organic solvents.

Molecular design of seed storage proteins for enhanced food physicochemical properties.

Seed storage proteins such as soybean globulins have been nutritionally and functionally valuable in the food industry. Protein structure-function studies are valuable in modifying proteins for enhanced functionality. Recombinant technology and protein engineering are two of the tools in biotechnology that have been used in producing soybean proteins with better gelling property, solubility, and emulsifying ability. This article reviews the molecular basis for the logical and precise protein designs that are important in obtaining the desired improved physicochemical properties.

Structure of 8Sα globulin, the major seed storage protein of mung bean

The 8S globulins of mung bean [Vigna radiata (L.) Wilczek] are vicilin-type seed storage globulins which consist of three isoforms: 8Sα, 8Sα′ and 8Sβ. The three isoforms have high sequence identities with each other (around 90%). The structure of 8Sα globulin has been determined for the first time by X-ray crystallographic analysis and refined at 2.65 A resolution with a final R factor of 19.6% for 10–2.65 A resolution data. The refined 8Sα globulin structure consisted of 366 of the 423 amino-acid residues (one subunit of the biological trimer). With the exception of several disordered regions, the overall 8Sα globulin structure closely resembled those of other seed storage 7S globulins. The 8Sα globulin exhibited the highest degree of sequence identity (68%) and structural similarity (a root-mean-square deviation of 0.6 A) with soybean β-conglycinin β (7S globulin). Their surface hydrophobicities are also similar to each other, although their solubilities differ under alkaline conditions at low ionic strength. This difference seems to be a consequence of charge–charge interactions and not hydrophobic interactions of the surfaces, based on a comparison of the electrostatic potentials of the molecular surfaces. The thermal stability of 8Sα globulin is lower than that of soybean β-conglycinin β. This correlates with the cavity size derived from the crystal structure, although other structural features also have a small effect on the proteins thermal stability.

Towards transformation, regeneration and screening of papaya containing antisense ACC synthase gene

The papaya (Carica papaya L.) is usually being harvested when they are about 25% ripe or when a tinge of yellow color appears on the skin. It takes only one to two weeks before the fruit ripens completely from the time of harvest. Losses due to postharvest diseases in some species reach up to about 60% of annual production. The two most important qualities required for efficient marketing of fruits are the taste and the overall appearance. The flexibility in marketing is determined mainly by the rate of fruit ripening. An extended ripening phase would prolong its shelf-life, thus allowing the fruit to be shipped to distant markets without spoilage, thereby increasing the potential target markets, and enabling the fruit to reach the market in better condition. The papaya is a climacteric fruit and ripening is in part, being controlled by the simple hydrocarbon ethylene. The rate-limiting enzyme of the ethylene biosynthetic pathway is ACC synthase. This enzyme has been the target in engineering the ethylene biosynthesis pathway using the antisense technology. This technology for instance has been used to reduce translation of either ACC synthase or ACC oxidase by antisense RNA that blocks ethylene production in tomato, thereby delaying fruit ripening (Oeller et al., 1991; Hamilton et al., 1991). Two ACC synthase genes expressed during fruit ripening have been cloned from papaya (Mason and Botella, 1997). These genes could be used for transforming papaya plants that could constitutively express an antisense copy of the ACC synthase gene. In this study, we aimed to apply antisense technology and the existing transformation protocol to produce transgenic Solo papayas.

Cloning, molecular analysis, and developmental expression of 3 oleosin cDNA isoforms in coconut (Cocos nucifera L.)

ABSTRACT Coconut stores its lipid reserves in the endosperm, specifically in oil bodies that contain proteins called oleosins which stabilise their structure. This study reports the complete cDNA sequences of 3 genes for 3 isoforms of oleosin termed CnOLE500a, CnOLE500c, and CnOLE300a with 396, 375, and 381 nucleotides, respectively. Their predicted amino acid sequences were 131, 124, and 126 residues in length, respectively, with molecular weights of 13,787, 12,982, and 12,988 Da, respectively. The complete CnOLE500a cDNA sequence had 83.1% similarity with that of CnOLE500c, while CnOLE300a cDNA had only 50.7% and 46.5% similarity with CnOLE500a and CnOLE500c, respectively. None of the 3 coconut oleosins had the 18 amino acid insertion characteristics of H-class oleosins, thus they belonged to the L-class of oleosins. However, phylogenetic analysis showed that CnOLE300a was more related to H-class oleosins from dicots. All 3 isoforms were highly expressed at all stages of coconut endosperm development. CnOLE500c exhibited 6% higher expression than CnOLE500a and 15% higher than CnOLE300a at all stages of coconut endosperm development. However, oleosin proteins were barely detectable in the solid endosperm of coconut at 5–6 months, but this increased 20-fold at 6–7 months, and increased by a further 2- and 12-fold, at 7–8 and 8–9 months, respectively.

Biochemical and molecular characterization of two 11S globulin isoforms from coconut and their expression analysis during seed development

Cocosin, the 11S globulin of coconut, is the major storage protein that accumulates in the endosperm during seed development. This work describes the gene structure, derived amino acid sequence, structural homologies, and developmental expression profile of cocosin. Two full-length cocosin cDNAs spanning 1,641 and 1,623 nucleotides and encoding for 466 amino acids were isolated through PCR cloning strategy coupled with 5′ and 3′-RACE technologies. Both sequences displayed 92.3% nucleotide identity and 91.5% amino acid identity. They both exhibit the following conserved regions among 11S globulins/ glutelins of various seed plants―signal peptide targeting storage vacuole deposition, highly conserved asparaginyl splice site dividing the polypeptides into acidic (32 kDa) and basic (21 kDa) subunits, bicupin domain and four cysteine residues involved in intraand inter-chain disulfide bonding. Phylogenetic analysis showed that cocosin is more closely related to Elaeis guineensis glutelin, both of which form a distinct clade in the tree between the divergent clades of dicot and cereal 11S globulins. The predicted three-dimensional structure of the cocosin exhibits the bicupin domain separated by a less conserved loop. Relative PCR and western blot analysis showed that synthesis of cocosin started at 6–7 months after pollination (MAP) and increased continuously up to 8–9 MAP. Western blot analysis further showed that majority of cocosin was deposited at 11–12 MAP. Keywords—cocosin, 11S globulins, Cocos nucifera L., storage protein, seed maturation, developmental expression