The Effects Of Pyruvate Kinase OsPK1on Important Agronomic Traits Of Rice And The Mechanism Research

Rice is an important cereal crop. How to increase rice yield and cultivate newvarieties which has favorable agronomic traits is a major issue. Rice is a modelplant of genome research in monocots. Rice research has entered the "functionalgenomics era." Pyruvate kinase (PK) is a key enzyme of the glycolytic pathway,and its effects on morphological development of rice are rarely reported. In thisstudy, we explored the effects of a cytosolic PK (PKc), OsPK1, on rice growthand development and the mechanism in them by identification of a rice T-DNAinsertion dwarf mutant ospk1and functional analysis of the gene OsPK1.The main phenotypes of mutant ospk1are dwarf, panicle enclosure andreduced seed set. Analysis of the flanking sequences revealed that a T-DNAintegration occurred at nucleotide position349bp upstream of the ATG startcodon of OsPK1. Quantitative RT-PCR analysis showed that the expression ofOsPK1was decreased by approximately90%in the root, leaf, internode, andpanicle of ospk1, which suggested that the T-DNA insertion site is in thetranscriptional regulatory region of OsPK1. OsPK1full-length genomicfragment was introduced into ospk1mutant to obtain transgenic plants forcomplementation. The phenotype of these transgenic plants totally reverted tothat of WT, which was judged from whether panicle is out of sheath, plantheight, seed set and seed germination. This confirmed that the OsPK1mutationis responsible for the ospk1phenotype. Subcellular localization and enzymeassay revealed that OsPK1encodes a PKc. Quantitative RT-PCR analysisshowed altered expression of four putative enzyme genes ofglycolysis/gluconeogenesis pathway. RT-PCR analysis showed that theexpression of Os01g0208700, a PEP carboxylase, was increased in ospk1. Thesesuggest that the decreased expression of OsPK1leads to altered expression ofcertain enzymes involved in related metabolic pathways. The expression patternof OsPK1was analyzed using quantitative RT-PCR, GUS staining andmicrotome sections. The highest level of OsPK1expression was in leaves. Inyoung seedlings, high levels of OsPK1expression were observed in coleoptile, the maturation zone of the radicle and adventitious roots. OsPK1expression wasalso detected in young floret and milky stage seeds. At the cellular level, OsPK1expression was localized in mesophyll cells in leaves, phloem companion cellsin vascular bundles in stems, and in cortical parenchyma cells in roots. Thus,OsPK1appears to be expressed predominately in these functional tissues andcell types, implying that OsPK1is critical for diverse metabolic processes in rice.Using LC/MS, we found that the contents of GA₄in the young sheath and GA₁in the uppermost internode (UI) were decreased in ospk1, and their precursorsaccumulated abundantly. We infer that the suppressed synthesis of bioactiveGAs in ospk1is likely to be one of the factors resulting in its dwarf phenotype.In addition, ospk1showed increased ABA contents in the two tissues, resultingin the variation in the ABA/GA balance. The contents of glucose, fructose,galactose and sucrose in different organs of WT and ospk1were measured usingGC/MS. These results indicated that the decreased expression of OsPK1haseffects on the biosynthesis, catabolism and transport of sugar, as well asexogenous sucrose (nutrient) uptake by root.This study clarified the effects of a PKc, OsPK1, on the important agronomictraits (plant height and seed set) and provided new insights into molecularmechanisms of plant height and seed set. OsPK1has very close orthologs inArabidopsis thaliana, maize and sorghum. Therefore, our work is a valuablereference to future study on those orthologs.