Hongzheng Sun

Characterization and Expression Patterns of microRNAs Involved in Rice Grain Filling

MicroRNAs (miRNAs) are upstream gene regulators of plant development and hormone homeostasis through their directed cleavage or translational repression of the target mRNAs, which may play crucial roles in rice grain filling and determining the final grain weight and yield. In this study, high-throughput sequencing was performed to survey the dynamic expressions of miRNAs and their corresponding target genes at five distinct developmental stages of grain filling. In total, 445 known miRNAs and 45 novel miRNAs were detected with most of them expressed in a developmental stage dependent manner, and the majority of known miRNAs, which increased gradually with rice grain filling, showed negatively related to the grain filling rate. Detailed expressional comparisons revealed a clear negative correlation between most miRNAs and their target genes. It was found that specific miRNA cohorts are expressed in a developmental stage dependent manner during grain filling and the known functions of these miRNAs are involved in plant hormone homeostasis and starch accumulation, indicating that the expression dynamics of these miRNAs might play key roles in regulating rice grain filling.

Differentially expressed microRNA cohorts in seed development may contribute to poor grain filling of inferior spikelets in rice

BackgroundThe inferior spikelets are defined to be those at portions where the grains receive less photosynthetic products during the seed development. The typical inferior spikelets are physically located on the proximal secondary branches in a rice panicle and traditionally characterized by a later flowering time and a slower grain-filling rate, compared to those so-called superior spikelets. Grains produced on the inferior spikelets are consequently under-developed and lighter in weight than those formed on the superior spikelets. MicroRNAs (miRNAs) are recognized as key players in regulating plant development through post-transcriptional gene regulations. We previously presented the evidence that miRNAs may influence grain-filling rate and played a role in determining the grain weight and yield in rice.ResultsIn this study, further analyses of the expressed small RNAs in superior and inferior spikelets were conducted at five distinct developmental stages of grain development. Totally, 457 known miRNAs and 13 novel miRNAs were analyzed, showing a differential expression of 141 known miRNAs between superior and inferior spikelets with higher expression levels of most miRNAs associated with the superior than the inferior spikelets during the early stage of grain filling. Genes targeted by those differentially expressed miRNAs (i.e. miR156, miR164, miR167, miR397, miR1861, and miR1867) were recognized to play roles in multiple developmental and signaling pathways related to plant hormone homeostasis and starch accumulation.ConclusionsOur data established a complicated link between miRNA dynamics and the traditional role of hormones in grain filling and development, providing new insights into the widely accepted concepts of the so-called superior and inferior spikelets in rice production.

qRT9, a quantitative trait locus controlling root thickness and root length in upland rice

Breeding for strong root systems is an important strategy for improving drought avoidance in rice. To clone genes responsible for strong root traits, an upland rice introgression line IL392 with thicker and longer roots than the background parent lowland rice Yuefu was selected. A quantitative trait locus (QTL), qRT9, controlling root thickness and root length was detected under hydroponic culture using 203 F(2:3) populations derived from a cross between Yuefu and IL392. The qRT9 locus explained 32.5% and 28.1% of the variance for root thickness and root length, respectively. Using 3185 F2 plants, qRT9 was ultimately narrowed down to an 11.5 kb region by substitution mapping. One putative basic helix-loop-helix (bHLH) transcription factor gene, LOC_Os09g28210 (named OsbHLH120), is annotated in this region. Sequences of OsbHLH120 in 11 upland rice and 13 lowland rice indicated that a single nucleotide polymorphism (SNP) at position 82 and an insertion/deletion (Indel) at position 628-642 cause amino acid changes and are conserved between upland rice and lowland rice. Phenotypic analysis indicated that the two polymorphisms were significantly associated with root thickness and root length under hydroponic culture. Quantitative real-time PCR showed that OsbHLH120 was strongly induced by polyethylene glycol (PEG), salt, and abscisic acid, but higher expression was present in IL392 roots than in Yuefu under PEG and salt stress. The successfully isolated locus, qRT9, enriches our knowledge of the genetic basis for drought avoidance and provides an opportunity for breeding drought avoidance varieties by utilizing valuable genes in the upland rice germplasm.

Genome-wide analysis of 24-nt siRNAs dynamic variations during rice superior and inferior grain filling.

24 nt-siRNAs are the most abundant small interfering RNAs in rice grains aside from microRNAs. To investigate the roles that 24 nt-siRNAs played in the poor grain filling of rice inferior grains, dynamic variations of 24 nt-siRNAs in inferior grains were compared with those of superior grains by using small RNA deep sequencing technology. The results showed that 24 nt-siRNAs derived from multiple regions of rice genome, and the maintenance of the two strands of 24 nt-siRNA duplex was a non-random process. The amounts of 24 nt-siRNAs declined with the process of grain filling in both superior and inferior grains, but 24 nt-siRNAs in inferior grains was much higher than that of superior grains in each period we sampled. Bioinformatics prediction indicated that 24 nt-siRNAs targeted on more genes involved in most of the known KEGG rice pathways, such as the starch and sucrose biosynthesis pathway. Combined with digital gene expression profiling of target genes, 24 nt-siRNAs mapped on the antisense strands of exons were specifically investigated, but the abundance of 24 nt-siRNAs did not show negative correlations with their corresponding target genes. The results indicated that 24 nt-siRNAs were not involved in down-regulation of target genes. The potential biological meanings for this inconsistency were probably the results of methylation directed gene expression activation, or competition for small RNA stability methylation.

Superior grains determined by grain weight are not fully correlated with the flowering order in rice

Rice panicles are composed of many branches with two types of extreme grains,the superior and the inferior.Traditionally,it has been well accepted that earlier flowers result in superior grains and late flowers generate inferior grains.However,these correlations have never been strictly examined in practice.In order to determine the accurate relationship between superior and inferior grains and the flowering order,we localized all the seeds in a panicle in four distinct rice species and systematically documented the rice flowering order,flower locations and the final grain weight for their relationships.Our results demonstrated that the grain weight is more heavily determined by the position of the seeds than by the flowering order.Despite earlier flowering has a positive correlation with the grain weight in general,grains from flowers blooming on the second day after anthesis generally gained the highest weight.This suggests earlier flowers may not result in superior grains.Therefore,we concluded that superior and inferior grains,commonly determined by grain weight,are not fully correlated with the flowering order in rice.Following the order of the grain weight,the superior grains are generally localized at the middle parts of the primary branches,whereas inferior grains were mainly on the last two secondary branches of the lower half part of the panicle.In addition,the weight of inferior grains were affected by spikelet thinning and spraying with exogenous plant growth regulators,indicating that physiological incompetence might be the major reason for the occurrence of the inferior grains.

Suppression of microRNA159 impacts multiple agronomic traits in rice ( Oryza sativa L . )

BackgroundmicroRNAs (miRNAs) are important regulators in plant growth and development. miR159 is a conserved miRNA among different plant species and has various functions in plants. Studies on miR159 are mostly done on model plant, Arabidopsis thaliana. In rice, studies on miR159 were either based upon genome-wide expression analyses focused upon responses to different nitrogen forms and abiotic stress or upon phenotypic studies of transgenic plants overexpressing its precursor. STTM (Short Tandem Target Mimic) is an effective tool to block the activity of endogenous mature miRNA activity in plant. Therefore, specific roles of miR159 in rice could be explored by down regulating miR159 through STTM.ResultsIn this study, expression of mature miR159 was successfully suppressed by STTM which resulted in the increased expressions of its two targets genes, OsGAMYB and OsGAMYBL1 (GAMYB-LIKE 1). Overall, STTM159 plants exhibited short stature along with smaller organ size and reduction in stem diameter, length of flag leaf, main panicle, spikelet hulls and grain size. Histological analysis of stem, leaf and mature spikelet hull showed the reduced number of small vascular bundles (SVB), less number of small veins (SV) between two big veins (LV) and less cell number in outer parenchyma. Gene Ontology (GO) enrichment analysis of differentially expressed genes between wild type plants and STTM159 transgenic plants showed that genes involved in cell division, auxin, cytokinin (CK) and brassinosteroids (BRs) biosynthesis and signaling are significantly down-regulated in STTM159 plants.ConclusionOur data suggests that in rice, miR159 positively regulates organ size, including stem, leaf, and grain size due to the promotion of cell division. Further analysis from the RNA-seq data showed that the decreased cell divisions in STTM159 transgenic plants may result, at least partly from the lower expression of the genes involved in cell cycle and hormone homeostasis, which provides new insights of rice miR159-specific functions.

Dynamic Analysis of Gene Expression in Rice Superior and Inferior Grains by RNA-Seq.

Poor grain filling of inferior grains located on lower secondary panicle branch causes great drop in rice yield and quality. Dynamic gene expression patterns between superior and inferior grains were examined from the view of the whole transcriptome by using RNA-Seq method. In total, 19,442 genes were detected during rice grain development. Genes involved in starch synthesis, grain storage and grain development were interrogated in particular in superior and inferior grains. Of the genes involved in sucrose to starch transformation process, most were expressed at lower level in inferior grains at early filling stage compared to that of superior grains. But at late filling stage, the expression of those genes was higher in inferior grains and lower in superior grains. The same trends were observed in the expression of grain storage protein genes. While, evidence that genes involved in cell cycle showed higher expression in inferior grains during whole period of grain filling indicated that cell proliferation was active till the late filling stage. In conclusion, delayed expression of most starch synthesis genes in inferior grains and low capacity of sink organ might be two important factors causing low filling rate of inferior grain at early filling stage, and shortage of carbohydrate supply was a limiting factor at late filling stage.

Influence of water potential and soil type on conventional japonica super rice yield and soil enzyme activities

Abstract We carried out a pool culture experiment to determine the optimal water treatment depth in loam and clay soils during the late growth stage of super rice. Three controlled water depth treatments of 0–5, 0–10 and 0–15 cm below the soil surface were established using alternate wetting and drying irrigation, and the soil water potential (0 to –25 kPa) was measured at 5, 10 and 15 cm. A 2-cm water layer was used as the control. We measured soil enzyme activities, root antioxidant enzyme activities, chlorophyll fluorescence parameters, and rice yield. The results showed that the 0–5-cm water depth treatment significantly increased root antioxidant enzyme activities in loam soil compared with the control, whereas soil enzyme activities, chlorophyll fluorescence parameters and yield did not differ from those of the control. The 0–10- and 0–15-cm water depth treatments also increased root antioxidant enzyme activities, whereas soil enzyme activities, chlorophyll fluorescence parameters and yield decreased. In clay soil, the soil enzyme activities, root antioxidant enzyme activities, chlorophyll fluorescence parameters, and yield did not change with the 0–5-cm water treatment, whereas the 0–10- and 0–15-cm water treatments improved these parameters. Therefore, the appropriate depths for soil water during the late growth period of rice with a 0 to –25 kPa water potential were 5 cm in loam and 15 cm in clay soil.

miR1432-OsACOT (Acyl-CoA thioesterase) module determines grain yield via enhancing grain filling rate in rice.

Summary Rice grain filling rate contributes largely to grain productivity and accumulation of nutrients. MicroRNAs (miRNAs) are key regulators of development and physiology in plants and become a novel key target for engineering grain size and crop yield. However, there is little studies, so far, showing the miRNA regulation of grain filling and rice yield, in consequence. Here, we show that suppressed expression of rice miR1432 (STTM1432) significantly improves grain weight by enhancing grain filling rate and leads to an increase in overall grain yield up to 17.14% in a field trial. Molecular analysis identified rice Acyl‐CoA thioesterase (OsACOT), which is conserved with ACOT13 in other species, as a major target of miR1432 by cleavage. Moreover, overexpression of miR1432‐resistant form of OsACOT (OXmACOT) resembled the STTM1432 plants, that is, a large margin of an increase in grain weight up to 46.69% through improving the grain filling rate. Further study indicated that OsACOT was involved in biosynthesis of medium‐chain fatty acids. In addition, RNA‐seq based transcriptomic analyses of transgenic plants with altered expression of miR1432 demonstrated that downstream genes of miR1432‐regulated network are involved in fatty acid metabolism and phytohormones biosynthesis and also overlap with the enrichment analysis of co‐expressed genes of OsACOT, which is consistent with the increased levels of auxin and abscisic acid in STTM1432 and OXmACOT plants. Overall, miR1432‐OsACOT module plays an important role in grain filling in rice, illustrating its capacity for engineering yield improvement in crops.

Difference of Bleeding Intensity in Different Parts of Rice Plant and Its Relationship with Grain Plumpness

This study aimed to illustrate the mechanism of inferior grain plumpness in rice. A field experiment with 0, 90 and 180 kg ha1 N spike fertilizer application in the stage of rice young ear differentiation was carried out in 2005 and 2006 on the farm of Henan Agricultural University, Henan Province, China. Two varieties (indica and japonica rice) were used to study the periodical changes of basal and neck internode bleeding intensity, soluble sucrose in bleeding, daily change of bleeding intensity and the relationship between the bleeding intensity and soluble sucrose of neck internode with grain plumpness in the grain filling stage. The results showed that the changes of bleeding intensity between the basal and neck internodes were obviously different. The bleeding intensity in the basal internode reached the maximum value in the fifth day after anthesis and presented the drop tendency afterwards. The bleeding intensity of the neck internode appeared two peak values in the fifth and seventeenth days after anthesis. There was a valley value in the eleventh day after anthesis on average. The average bleeding intensity in the neck internode was higher under zero N lever than high N level. The daily change of bleeding intensity in the neck internode was low in the daytime and high at night, which reduced with rising N fertilizer application. Correlation analysis showed that there existed significantly positive correlation or highly signification between inferior grain plumpness and the bleeding intensity of neck internode in the 10, 15, 25, and 35 days after anthesis for the japonica, but not for the indica rice. The relationship between solublesucrose in the bleeding of neck internode and inferior grain plumpness showed highly significantly positive correlation, while that of the basal internode was not notable. It indicated that the relationship between bleeding in the neck internode and the inferior grain plumpness is close. The bleeding intensity of neck internode can be used as a index for measuring inferior grain plumpness. Therefore, increasing the bleeding intensity of neck internode by regulating N application can improve the inferior grain plumpness.