Yubi Huang

Analysis of synonymous codon usage in Zea mays

It is important and meaningful to understand the codon usage pattern and the factors that shape codon usage of maize. In this study, trends in synonymous codon usage in maize have been firstly examined through the multivariate statistical analysis on 7402 cDNA sequences. The results showed that the genes positions on the primary axis were strongly negatively correlated with GC3s, GC content of individual gene and gene expression level assessed by the codon adaptation index (CAI) values, which indicated that nucleotide composition and gene expression level were the main factors in shaping the codon usage of maize, and the variation in codon usage among genes may be due to mutational bias at the DNA level and natural selection acting at the level of mRNA translation. At the same time, CDS length and the hydrophobicity of each protein were, respectively, significantly correlated with the genes locations on the primary axis, GC3s and CAI values. We infer that genes length and the hydrophobicity of the encoded protein may play minor role in shaping codon usage bias. Additional 28 codons ending with a G or C base have been defined as “optimal codons”, which may provide useful information for maize gene-transformation and gene prediction.

Synergistic influence of sucrose and abscisic acid on the genes involved in starch synthesis in maize endosperm

Starch is the major carbon reserve in plant storage organs, the synthesis of which is orchestrated by four major enzymes, ADP-glucose pyrophosphorylase, starch synthase, starch-branching enzyme and starch-debranching enzyme. There is much information available on the function of these key enzymes; however, little is known about their transcriptional regulation. In order to understand the transcriptional regulation of starch biosynthesis, the expression profiles of 24 starch genes were investigated in this work. The results showed major transcriptional changes for 15 of the 24 starch genes observed in maize endosperm, most of which are elevated at the early and middle stages of the developing endosperm. Sucrose, abscisic acid (ABA) and indole-3-acetic acid (IAA) had a significant correlation with the expression of 15 genes, indicating that sugars and phytohormones might take part in the regulation of starch synthesis. Also, we found that there is interaction of abscisic acid and sucrose on the regulation of the expression of these genes.

Binding of ABI4 to a CACCG motif mediates the ABA-induced expression of the ZmSSI gene in maize (Zea mays L.) endosperm

Starch synthase I (SSI) contributes the majority of the starch synthase activity in developing maize endosperm. In this work, the effects of various plant hormones and sugars on the expression of the starch synthase I gene (ZmSSI) in developing maize endosperms were examined. The accumulation of ZmSSI mRNA was induced using abscisic acid (ABA) but not with glucose, sucrose, or gibberellin treatment. To investigate the molecular mechanism underlying this effect, the ZmSSI promoter region (-1537 to +51) was isolated and analysed. A transient expression assay in maize endosperm tissue showed that the full-length ZmSSI promoter is activated by ABA. The results of deletion and mutation assays demonstrated that a CACCG motif in the ZmSSI promoter is responsible for the ABA inducibility. The results of binding shift assays indicated that this CACCG motif interacts with the maize ABI4 protein in vitro. The overexpression of ABI4 in endosperm tissue enhanced the activity of a promoter containing the CACCG motif in the absence of ABA treatment. Expression pattern analysis indicated that the transcription pattern of ABI4 in the developing maize endosperm was induced by ABA treatment but was only slightly affected by glucose or sucrose treatment. Taken together, these data indicate that ABI4 binds to the CACCG motif in the ZmSSI promoter and mediates its ABA inducibility.

Novel role of ZmaNAC36 in co-expression of starch synthetic genes in maize endosperm

Abstract Starch is an essential commodity that is widely used as food, feed, fuel and in industry. However, its mechanism of synthesis is not fully understood, especially in terms of the expression and regulation of the starch synthetic genes. It was reported that the starch synthetic genes were co-expressed during maize endosperm development; however, the mechanism of the co-expression was not reported. In this paper, the ZmaNAC36 gene was amplified by homology-based cloning, and its expression vector was constructed for transient expression. The nuclear localization, transcriptional activation and target sites of the ZmaNAC36 protein were identified. The expression profile of ZmaNAC36 showed that it was strongly expressed in the maize endosperm and was co-expressed with most of the starch synthetic genes. Moreover, the expressions of many starch synthesis genes in the endosperm were upregulated when ZmaNAC36 was transiently overexpressed. All our results indicated that NAC36 might be a transcription factor and play a potential role in the co-expression of starch synthetic genes in the maize endosperm.

A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes

Key messageConditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein–protein interaction, and the investigation of PCD-related processes.AbstractPlant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein–protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.

Identification and Phylogenetic Analysis of a Novel Starch Synthase in Maize

Starch is an important reserve of carbon and energy in plants, providing the majority of calories in the human diet and animal feed. Its synthesis is orchestrated by several key enzymes, and the amount and structure of starch, affecting crop yield and quality, are determined mainly by starch synthase (SS) activity. To date, five SS isoforms, including SSI-IV and Granule Bound Starch Synthase (GBSS) have been identified and their physiological functions have been well characterized. Here, we report the identification of a new SS isoform in maize, designated SSV. By searching sequenced genomes, SSV has been found in all green plants with conserved sequences and gene structures. Our phylogenetic analysis based on 780 base pairs has suggested that SSIV and SSV resulted from a gene duplication event, which may have occurred before the algae formation. An expression profile analysis of SSV in maize has indicated that ZmSSV is mainly transcribed in the kernel and ear leaf during the grain filling stage, which is partly similar to other SS isoforms. Therefore, it is likely that SSV may play an important role in starch biosynthesis. Subsequent analysis of SSV function may facilitate understanding the mechanism of starch granules formation, number and structure.

Identification and characterization of microRNAs in the developing maize endosperm

MicroRNAs (miRNAs) are non-coding RNAs that are approximately 20-22 nucleotides long. miRNAs have been shown to be important regulators that control a large variety of biological functions in eukaryotic cells. To investigate the roles of miRNAs in maize endosperm development, two small RNA libraries of maize endosperm at two developmental stages were sequenced. A total of 17,773,394 and 18,586,523 small RNA raw reads were obtained, respectively. Further analysis identified and characterized 95 known miRNAs belonging to 20 miRNA families. In addition, 18 novel miRNAs were identified and grouped into 11 families. Potential targets for 5 of the novel miRNA families were successfully predicted. We had also identified 12 corresponding miRNAs* of these novel miRNAs. In summary, we investigated expression patterns of miRNA in maize endosperm at key developmental stages and identified miRNAs that are likely to playing an important role in endosperm development.

Characterization of ADP-Glucose Pyrophosphorylase Encoding Genes in Source and Sink Organs of Maize

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis. Six cDNA sequences encoding three large subunits and three small subunits of maize AGPase from database were mined and subsequently named: agpl1, agpl2, agpl3, agps1, agps2, and agps3. To elucidate the roles of these isogenes, a comprehensive expression analysis of the gene family was conducted by quantitative real-time RT-PCR. Based on the expression patterns, the six genes can be divided into three groups: (1) steady expressers (agpl1, agps1, and agpl2), which were expressed relatively constantly both in leaf and grain; (2) tissue and development-specific expressers (agpl3 and agps2), which were expressed only in grain at middle and late development phases; (3) tissue-specific expressers (agps3), whose transcripts kept constant during grain filling and were observed only in grain. In order to clarify the effects of sugar and plant hormone on maize AGPase genes expression, a serial of treatments were used. The results showed that AGPase genes significantly differed in response to sugar and hormone inductions. Enormous transcript changes of these genes could be observed in glucose and sucrose treatments. Interestingly, synergistic effect of ABA and sucrose on these genes was observed.

Waxy locus and its mutant types in maize Zea mays L.

The waxy (wx) locus of Zea mays L. encodes a granule-bound starch synthase (GBSS I=waxy protein) required for the synthesis of amylose in endosperm and pollen grain. This review covers recent advances in understanding the waxy locus in maize, focusing particularly on the new information on mutant type and mutation mechanisms. The results showed that the insertion and deletion played an important role in the generation of spontaneous wx-mutations. The current status of utilizing waxy locus has been summarized and the perspectives of the further studies on this locus have also been proposed.

Relationship between activities of key enzymes involved in starch synthesis and accumulation in maize inbred lines during grain filling

Time course of starch production and the key enzyme activities in the grains of four maize inbred lines (two high-starch and two low-starch lines) were studied. Accumulation of grain starch and its components in four maize inbred lines rose continuously after pollination and increased as a sigmoid curve during grain filling. The accumulation rates showed single-peak curves. The accumulation rates of starch and its components reached their peaks on 25–32 days after pollination (DAP), respectively. Activities of adenosine diphosphoglucose pyrophosphorylase (AGPPase) and starch synthase in the grains of four lines followed single-peak curves with the peaks on 24–31 DAP. The highest activity of the starch-branching enzyme (Q-enzyme) in the grains of both high-starch lines appeared on 23 DAP, but that of both low-starch inbred lines showed double-peak curves, the peaks being at 15–20 DAP and 30–35 DAP. There was significant positive correlation between AGPPase, soluble starch synthase (SSS), and starch granule-bound synthase (GBSS) activities. The results indicated the Q-enzyme had different expression patterns in the high-and the low-starch maize inbred lines, and that AGPPase, SSS, and GBSS activities were significantly and positively correlated with amylose, amylopectin, and starch accumulation rates in all lines.