Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor

Suc, an end product of photosynthesis, is metabolized by Suc synthase in sink organs as an initial step in the biosynthesis of storage products. Suc synthase activity is known to be regulated by reversible phosphorylation, but the details of this process are unclear at present. Rice SPK, a calcium-dependent protein kinase, is expressed uniquely in the endosperm of immature seed, and its involvement in the biosynthetic pathways of storage products was suggested. Antisense SPK transformants lacked the ability to accumulate storage products such as starch, but produced watery seed with a large amount of Suc instead, as the result of an inhibition of Suc degradation. Analysis of in vitro phosphorylation indicated that SPK phosphorylated specifically a Ser residue in Suc synthase that has been shown to be important for its activity in the degradation of Suc. This finding suggests that SPK is involved in the activation of Suc synthase. It appears that SPK is a Suc synthase kinase that may be important for supplying substrates for the biosynthesis of storage products.     

Evaluation of allergenic potential for rice seed protein components utilizing a rice proteome database and an allergen database in combination with IgE-binding of recombinant proteins

Among 131 rice endosperm proteins previously identified by MS-based proteomics, most of the proteins showed low or almost no sequence similarity to known allergens in databases, whereas nine proteins did it significantly. The sequence of two proteins showed high overall identity with Hsp70-like hazel tree pollen allergen (Cor a 10) and barley α-amylase (Hor v 16), respectively, whereas the others showed low identity (28–58%) with lemon germin-like protein (Cit l 1), corn zein (Zea m 50 K), wheat chitinase-like xylanase inhibitor (Tri a XI), and kinase-like pollen allergen of Russian thistle (Sal k 1). Immuno-dot blot analysis showed that recombinant proteins for these rice seed homologs were positive in the IgE-binding, but not necessarily similarity dependent, from some allergic patients. These results suggest that utilization of proteome and sequence databases in combination with IgE-binding analysis was effective to screen and evaluate allergenic potential of rice seed protein components.     

NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase,regulates innate immunity and development in plants

Mitogen-activated protein kinase (MAPK) cascades are rapidly activated upon plant recognition of invading pathogens. Here, we describe the use of virus-induced gene silencing (VIGS) to study the role of candidate plant MAP kinase kinase kinase (MAPKKK) homologs of human MEKK1 in pathogen-resistance pathways. We demonstrate that silencing expression of a tobacco MAPKKK, Nicotiana Protein Kinase 1 (NPK1), interferes with the function of the disease-resistance genes N, Bs2, and Rx, but does not affect Pto- and Cf4-mediated resistance. Further, NPK1-silenced plants also exhibit reduced cell size, defective cytokinesis, and an overall dwarf phenotype. Our results provide evidence that NPK1 functions in the regulation of N-, Bs2-, and Rx-mediated resistance responses and may play a role in one or more MAPK cascades, regulating multiple cellular processes.     

SMALL GRAIN 1, which encodes a mitogen‐activated protein kinase kinase 4, influences grain size in rice

Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map‐based cloning approach, in mitogen‐activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)–OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR‐related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth.     

FLOURY ENDOSPERM16 encoding a NAD‐dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.     

Ospapst1, a useful mutant for identifying seed purity and authenticity in hybrid rice

The stability and completeness of male sterility is still a challenge in some male sterile rice lines, especially those of photoperiod/thermo-sensitive genic male sterility (P/TGMS). Leaf color marker is a widely practiced approach to reduce the impact of self-pollinated seeds of male sterile lines. The papst1 is a leaf color mutant. The newly emerged leaves of papst1 are chlorosis and have an impaired photosynthesis. But the other agronomic traits, such as germination rate, duration of maturation and seed weight, are not changed. The papst1/PAPST1 F₁ showed the wild-type leaf phenotype. The papst1/PAPST1 F₂ progenies displayed an approximately 3:1 segregation ratio of WT phenotype:mutant phenotype (72: 28, χ² = 0.48, p > 0.05), suggesting that papst1 mutant phenotype is caused by a single repressive gene. Map-based cloning and sequencing analysis revealed that a point mutation was occurred in Os01 g16040 (OsPAPST1). Given these results, the Ospapst1 mutant is a useful mutant for identifying seed purity and authenticity in hybrid rice.