Analysis of Small RNAs Associated with Plant Senescence

Abstract

Senescence is an agriculturally significant process due to its negative impact to crop yield and postharvest quality. The genetic regulatory systems controlling senescence induction and progress respond to both developmental and environmental stress signals and involve numerous gene expression changes. Knowledge about the key molecular factors which control senescence is very limited. MicroRNAs (miRNAs) are a class of small RNAs which typically function by guiding cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes including development, responses to environmental stresses, and senescence. The long-term goal of this work is to elucidate roles of small RNAs associated with plant senescence. The hypothesis underlying this research is that miRNA-mediated regulation makes important contributions to the senescence process in plants. Specific, original research objectives included: 1) Profiling of small RNAs from senescing plants; 2) Data Analysis and public access via a user-friendly web interface; 3) Validation of senescence-associated miRNAs and target RNAs; 4) Development of transgenic plants for functional analysis of miRNAs in Arabidopsis. Major revisions made in the research compared to the original work plan included 1) Exclusion of the planned work with tomato as recommended by the BARD review panel; 2) Performing miRNA study also in senescing Arabidopsis siliques, in addition to senescing leaves. To identify senescenceregulation of miRNAs in Arabidopsis thaliana, eight small RNA libraries were constructed and sequenced at four different stages of development and senescence from both leaves and siliques, resulting in more than 200 million genome-matched sequences. Parallel Analysis of RNA Ends (PARE) libraries, which enable the large-scale examination of miRNA-guided cleavage products, were also constructed and sequenced, resulting in over 750 million genome-matched sequences. These massive datasets lead to the identification of new miRNAs, as well as new regulation of known miRNAs and their target genes during senescence, many of which have established roles in nutrient responsiveness and cell structural integrity. In keeping with remobilization of nutrients thought to occur during senescence, many miRNAs and targets had opposite expression pattern changes between leaf and silique tissues during the progression of senescence. Taken together, these findings highlight the integral role that miRNAs may play in the remobilization of resources and alteration of cellular structure that is known to occur in senescence. Experiments were initiated for functional analysis of specific senescence-associated miRNAs and respective target genes. Transgenic Arabidopsis plants were generated in which miR408, found in this study to be significantly induced in leaf senescence, was over-expressed either constitutively or under a senescence-specific promoter. These plants are currently being characterized for any altered phenotypes. In addition T-DNA knock out mutants for various target genes identified in this research are being analyzed. This work provides insights about specific miRNAs that contribute to leaf and silique senescence. The knowledge generated may suggest new strategies to monitor and alter the progression of senescence in crops for agricultural improvement.