Studies On The Mechanism Of Rudimentary Leaf Senescence In Litchi Panicle

Litchi (Litchi chinensis Sonn.) is an important evergreen fruit crop with high commercial value in tropical and subtropical regions. However, due to the environment and internal genetic factors, the yield of litchi is unstable and profoundly affected by the quality of flowers. The low quality of litchi flowers has been an important bottleneck constraining litchi industrial. Here, we explored whether and how the development of leaf on panicle (LoP) or rudimentary leaf (RL) affects the development of litchi panicle in two cultivated varieties (cv.) ’Nuomici’ and ’Geiwei’. Via reactive oxygen species (ROS) and low temperature treatment, we investigated the mechanism of premature leaf senescence in cv. ’Nuomici’ RNA samples extracted from different development stages of rudimentary leaves was analyzed by high-throughput sequencing. The gene expression data and transcriptome sequencing data analysis revealed that most NAC transcription factors and Metacaspase genes are related to LoP/RL senescence. The VIGS technology was used to study the function of these candidate genes furtherly. Our research investigated the underlying mechanism of LoP/RL senescence, elucidated the effects of LoP/RL senescence on panicle development, which surely shed light upon the molecular mechanism of litchi flowering. The main research results are as follows:(1) Through biological investigation, the different implications of leaf development on inflorescence growth between ’Nuomici’ and ’Guiwei’ were explored. Through an annual investigation, we found that the rudimentary leaf in ’Nuomici’ panicle was sensitive to either ROS or low temperature treatment. Under the stress of either ROS or low temperature, the leaf on ’Nuomici’ panicle could not develop normally and become premature senescent, but the flower buds developed normally. However, there was no obvious stress response of the leaf and flower buds in ’Guiwei’ group under the same treatment. Both the rudimentary leaf and flower buds grew and develop normally. The survey data from 2014 to 2015 showed that the existence of the rudimentary leaf seriously affected the development of flower bud on’Nuomici’, while there was no obvious difference on’Guiwei’.(2) Under the condition of greenhouse temperature control, potted’Nuomici’with "whitish millets" (panicle primordia) were placed in the low temperature (18/15℃癈) and high temperature (28/25℃) greenhouses separately. Different development stages of rudimentary leaves and flower buds were sampled and uesed for expression profile of sequencing. Through data analysis, leaf senescence associated genes were screened out. We found that NAC transcription factors were widely involved in this processes. The NAC transcription factors were choosed as the key research object for further analysis. The differentialy expressed genes (DEGs) were subjected to GO classifications, and DEGs in leaf samples were mainly classified into metabolic process, cellular process and protein metabolic process categories. The DEGs in flower buds samples were mainly classified into binding, cell and metabolic process categories.(3) We cloned the full length of LcNAC5, LcNAC4 and LcNAP1. The results of NCBI blastX showed that they belong to NAM superfamily. The homologous relationship of LcNAC5, LcNAC4, LcNAP1 and members in other plants was showed by homologous comparison and the evolutionary tree. RT-qPCR results showed that LcNAC5, LcNAC4 and LcNAP1 participated in the MV-induced rudimentary leaf senescence. The excised leaves of transgenic Arabidopsis thaliana and tobacoo plants overexpressing LcNAC5, LcNAC4 or LcNAP1 showed faster senescence under MV treatment compared to the nontransgenic control. These results show that LcNAC5, LcNAC4 and LcNAPl are involved in the ROS-induced senescence.(4) VIGS technology was used to investigate the function of LcNAC5. We used VIGS to silence LcNAC5 in the litchi rudimentary leaves, and MV was used to promote the leaf senescence. The results showed that the LcNAC5-silenced leaves manifested slowed senescence. The LcNAC5-silenced and control samples after 30 h of MV treatment were collected for RNA-seq sequencing. Associated candidate genes with LcNAC5 were filtered in this process, and 733 genes were up-regulated and 1857 genes were down-regulated. This data will be conducive to study genes interactions and leaf senescence mechanism.(5) Comprehensively analyze the previous results, we found that Metacaspase participated in litchi rudimentary leaf senescence. We cloned the full length of LcMC Ⅱ-1 and RT-qPCR results showed that it involved in litchi different stages of leaf senescence. We used VIGS to silence LcMC Ⅱ-1 in leaves of’Nuomici’, and the silenced leaves showed delayed senescence after MV treatment. The excised leaves of transgenic Arabidopsis plants overexpressing LcMC Ⅱ-1 under MV treatment showed faster senescence compared to the control. These results suggested that LcMC II-1 is involved in ROS-induced leaf senescence, and implicated that LcMC Ⅱ-1 plays an important role in litchi rudimentary leaf senescence.