UB2/UB3/TSH4-anchored transcriptional networks regulate early maize inflorescence development in response to simulated shade.

Increasing planting density has been adopted as an effective means to increase maize (Zea mays) yield. Competition for light from neighbors can trigger plant shade avoidance syndrome, which includes accelerated flowering. However, the regulatory networks of maize inflorescence development in response to high-density planting remain poorly understood. In this study, we showed that shade-mimicking treatments cause precocious development of the tassels and ears. Comparative transcriptome profiling analyses revealed the enrichment of phytohormone-related genes and transcriptional regulators among the genes co-regulated by developmental progression and simulated shade. Network analysis showed that three homologous Squamosa promoter binding protein (SBP)-like (SPL) transcription factors, Unbranched2 (UB2), Unbranched3 (UB3), and Tasselsheath4 (TSH4), individually exhibited connectivity to over 2,400 genes across the V3-to-V9 stages of tassel development. In addition, we showed that the ub2 ub3 double mutant and tsh4 single mutant were almost insensitive to simulated shade treatments. Moreover, we demonstrated that UB2/UB3/TSH4 could directly regulate the expression of Barren inflorescence2 (BIF2) and Zea mays teosinte branched1/cycloidea/proliferating cell factor30 (ZmTCP30). Furthermore, we functionally verified a role of ZmTCP30 in regulating tassel branching and ear development. Our results reveal a UB2/UB3/TSH4-anchored transcriptional regulatory network of maize inflorescence development and provide valuable targets for breeding shade-tolerant maize cultivars.

The evening complex promotes maize flowering and adaptation to temperate regions.

Maize (Zea mays) originated in southern Mexico and has spread over a wide latitudinal range. Maize expansion from tropical to temperate regions has necessitated a reduction of its photoperiod sensitivity. In this study, we cloned a quantitative trait locus (QTL) regulating flowering time in maize and show that the maize ortholog of Arabidopsis thaliana EARLY FLOWERING3, ZmELF3.1, is the causal locus. We demonstrate that ZmELF3.1 and ZmELF3.2 proteins can physically interact with ZmELF4.1/4.2 and ZmLUX1/2, to form evening complex(es; ECs) in the maize circadian clock. Loss-of-function mutants for ZmELF3.1/3.2 and ZmLUX1/2 exhibited delayed flowering under long-day and short-day conditions. We show that EC directly represses the expression of several flowering suppressor genes, such as the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) genes ZmCCT9 and ZmCCT10, ZmCONSTANS-LIKE 3, and the PSEUDORESPONSE REGULATOR (PRR) genes ZmPRR37a and ZmPRR73, thus alleviating their inhibition, allowing florigen gene expression and promoting flowering. Further, we identify two closely linked retrotransposons located in the ZmELF3.1 promoter that regulate the expression levels of ZmELF3.1 and may have been positively selected during postdomestication spread of maize from tropical to temperate regions during the pre-Columbian era. These findings provide insights into circadian clock-mediated regulation of photoperiodic flowering in maize and new targets of genetic improvement for breeding.

ZmELF3.1 integrates the RA2-TSH4 module to repress maize tassel branching.

Tassel branch number (TBN) is a key agronomic trait for adapting to high-density planting and grain yield in maize. However, the molecular regulatory mechanisms underlying tassel branching are still largely unknown. Here, we used molecular and genetic studies together to show that ZmELF3.1 plays a critical role in regulating TBN in maize. Previous studies showed that ZmELF3.1 forms the evening complex through interacting with ZmELF4 and ZmLUX to regulate flowering in maize and that RA2 and TSH4 (ZmSBP2) suppresses and promotes TBN in maize, respectively. In this study, we show that loss-of-function mutants of ZmELF3.1 exhibit a significant increase of TBN. We also show that RA2 directly binds to the promoter of TSH4 and represses its expression, thus leading to reduced TBN. We further demonstrate that ZmELF3.1 directly interacts with both RA2 and ZmELF4.2 to form tri-protein complexes that further enhance the binding of RA2 to the promoter of TSH4, leading to suppressed TSH4 expression and consequently decreased TBN. Our combined results establish a novel functional link between the ELF3-ELF4-RA2 complex and miR156-SPL regulatory module in regulating tassel branching and provide a valuable target for genetic improvement of tassel branching in maize.

Local auxin biosynthesis regulates brace root angle and lodging resistance in maize.

Root lodging poses a major threat to maize production, resulting in reduced grain yield and quality, and increased harvest costs. Here, we combined expressional, genetic, and cytological studies to demonstrate a role of ZmYUC2 and ZmYUC4 in regulating gravitropic response of the brace root and lodging resistance in maize. We show that both ZmYUC2 and ZmYUC4 are preferentially expressed in root tips with partially overlapping expression patterns, and the protein products of ZmYUC2 and ZmYUC4 are localized in the cytoplasm and endoplasmic reticulum, respectively. The Zmyuc4 single mutant and Zmyuc2/4 double mutant exhibit enlarged brace root angle compared with the wild-type plants, with larger brace root angle being observed in the Zmyuc2/4 double mutant. Consistently, the brace root tips of the Zmyuc4 single mutant and Zmyuc2/4 double mutant accumulate less auxin and are defective in proper reallocation of auxin in response to gravi-stimuli. Furthermore, we show that the Zmyuc4 single mutant and the Zmyuc2/4 double mutant display obviously enhanced root lodging resistance. Our combined results demonstrate that ZmYUC2- and ZmYUC4-mediated local auxin biosynthesis is required for normal gravity response of the brace roots and provide effective targets for breeding root lodging resistant maize cultivars.

miR169q and NUCLEAR FACTOR YA8 enhance salt tolerance by activating PEROXIDASE1 expression in response to ROS.

Salt stress significantly reduces the productivity of crop plants including maize (Zea mays). miRNAs are major regulators of plant growth and stress responses, but few studies have examined the potential impacts of miRNAs on salt stress responses in maize. Here, we show that ZmmiR169q is responsive to stress-induced ROS signals. After detecting that salt stress and exogenous H2O2 treatment reduced the accumulation of ZmmiR169q, stress assays with transgenic materials showed that depleting ZmmiR169q increased seedling salt tolerance whereas overexpressing ZmmiR169q decreased salt tolerance. Helping explain these observations, we found that ZmmiR169q repressed the transcript abundance of its target NUCLEAR FACTOR YA8 (ZmNF-YA8), and overexpression of ZmNF-YA8 in maize improved salt tolerance, specifically by transcriptionally activating the expression of the efficient antioxidant enzyme PEROXIDASE1. Our study reveals a direct functional link between salt stress and a miR169q-NF-YA8 regulatory module that plants use to manage ROS stress and strongly suggests that ZmNF-YA8 can be harnessed as a resource for developing salt-tolerant crop varieties.

miR169o and ZmNF-YA13 act in concert to coordinate the expression of ZmYUC1 that determines seed size and weight in maize kernels.

MicroRNAs (miRNAs) play key regulatory roles in seed development and emerge as new key targets for engineering grain size and yield. The Zma-miRNA169 family is highly expressed during maize seed development, but its functional roles in seed development remain elusive. Here, we generated zma-miR169o and ZmNF-YA13 transgenic plants. Phenotypic and genetic analyses were performed on these lines. Seed development and auxins contents were investigated. Overexpression of maize miRNA zma-miR169o increases seed size and weight, whereas the opposite is true when its expression is suppressed. Further studies revealed that zma-miR169 acts by negatively regulating its target gene, a transcription factor ZmNF-YA13 that also plays a key role in determining seed size. We demonstrate that ZmNF-YA13 regulates the expression of the auxin biosynthetic gene ZmYUC1, which modulates auxin levels in the early developing seeds and determines the number of endosperm cells, thereby governing maize seed size and ultimately yield. Overall, our present study has identified zma-miR169o and ZmNF-YA13 that form a functional module regulating auxin accumulation in maize seeds and playing an important role in determining maize seed size and yield, providing a set of novel molecular tools for yield improvement in molecular breeding and genetic engineering.

Interactions of Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN reveal a novel intracellular auxin transport mechanism.

Little is known about the transport mechanism of intracellular auxin. Here, we report two vacuole-localized proteins, Oryza sativa OsCONTINUOUS VASCULAR RING-LIKE 1 (OsCOLE1) and OsCOLE1-INTERACTING PROTEIN (OsCLIP), that regulate intracellular auxin transport and homoeostasis. Overexpression of OsCOLE1 markedly increased the internode length and auxin content of the stem base, whereas these parameters were decreased in RNA interference (RNAi) plants. OsCOLE1 was localized on the tonoplast and preferentially expressed in mature tissues. We further identified its interacting protein OsCLIP, which was co-localized on the tonoplast. Protein-protein binding assays demonstrated that the N-terminus of OsCOLE1 directly interacted with OsCLIP in yeast cells and the rice protoplast. Furthermore, (3) H-indole-3-acetic acid ((3) H-IAA) transport assays revealed that OsCLIP transported IAA into yeast cells, which was promoted by OsCOLE1. The results indicate that OsCOLE1 affects rice development by regulating intracellular auxin transport through interaction with OsCLIP, which provides a new insight into the regulatory mechanism of intracellular transport of auxin and the roles of vacuoles in plant development.

Advances on plant miR169/NF-YA regulation modules.

Plant microRNAs (miRNAs) are a class of small non-coding RNAs which target and regulate the expression of genes involved in several growth, development, and metabolism processes. The miR169 family is the largest and most conserved miRNA family in plants. Recent researches have shown involvement of miR169 members in the regulation of plant responding to abiotic stress and development. Computational prediction and experimental analyses suggest that miR169 targets members of the transcription factor NF-YA gene family. In this review, we summarize the origins, evolution and diversity of miR169 family members. In addition, a comprehensive understanding the regulatory functions of the miR169/NF-YA module in plant with particular emphasis on stress-induced flowering is discussed.

ZmGRF, a GA regulatory factor from maize, promotes flowering and plant growth in Arabidopsis.

Transcription factors that act as positive regulators of gibberellin (GA) biosynthetic genes in plants are not well understood. A nuclear-localized basic leucine zipper transcription factor, ZmGRF, was isolated from maize. The core DNA sequence motif recognized for binding by ZmGRF was CCANNTGGC. ZmGRF overexpression in transgenic Arabidopsis plants promoted flowering, stem elongation, and cell expansion. Chromatin immunoprecipitation assays revealed that ZmGRF bound directly to the cis-element CCANNTGGC in the promoter of the Arabidopsis ent-kaurene oxidase (AtKO1) gene and promoted AtKO1 expression. GA4 content increased by 372-567% in transgenic Arabidopsis plants overexpressing ZmGRF compared to wild-type control plants. The GIBBERELLIN-INSENSITIVE DWARF1 gene, which encodes a GA receptor, was also upregulated and the growth-repressing DELLA protein gene GA INSENSITIVE was downregulated. Our results showed ZmGRF functioned through the GA-signaling pathway.

ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis.

Construction of a genomewide RNAi mutant library in rice.

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification-mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down-regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double-stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross-silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA-derived siRNAs were characteristic of Argonaute-binding small RNAs. Our results indicate that RNAi mutant library is a high-efficient approach for genomewide gene identification in plants.

GmTMT2a from soybean elevates the α-tocopherol content in corn and Arabidopsis.

Tocochromanol, or vitamin E, plays a crucial role in human and animal nutrition and is synthesized only by photosynthetic organisms. γ-Tocopherol methyltransferase (γ-TMT), one of the key enzymes in the tocopherol biosynthetic pathway in plants, converts γ, δ-tocopherols into α-, ß-tocopherols. Tocopherol content was investigated in 15 soybean cultivars and GmTMT2 was isolated from five varieties based on tocopherol content. GmTMT2a was expressed in E. coli and the purified protein effectively converted γ-tocopherol into α-tocopherol in vitro. Overexpression of GmTMT2a enhanced α-tocopherol content 4-6-fold in transgenic Arabidopsis, and α-tocopherol content increased 3-4.5-fold in transgenic maize seed, which correlated with the accumulation of GmTMT2a. Transgenic corn that is α-tocopherol-rich may be beneficial for animal health and growth.

OsBSK3 Positively Regulates Grain Length and Weight by Inhibiting the Phosphatase Activity of OsPPKL1.

Brassinosteroids (BRs) are a crucial class of plant hormones that regulate many important agronomic traits in rice (Oryza sativa L.); thus, the BR signaling pathway is a very important tool for breeders to improve the grain yield and quantity of rice. Contrary to the well-established BR signaling pathway in Arabidopsis, there are significant gaps in the rice BR signaling pathway, especially the regulation mechanism from OsBSK3 to OsPPKLs and OsGSKs. In this study, we report how OsBSK3 knockout mutants confer shorter and lighter grains and exhibit a typical BR-insensitive phenotype, suggesting OsBSK3 plays a positive role in BR signaling without genetic redundancy with homologs. Furthermore, OsBSK3 could physically interact with OsPPKL1 and OsGSK3, the downstream components in BR signaling, as a scaffold protein, and inhibit the phosphatase activity of OsPPKL1 on the dephosphorylation of OsGSK3. In addition, the genetic evidence showed OsBSK3 acts upstream of OsPPKL1 in regulating grain length and weight. Our results clarify the role of OsBSK3 and provide new insights into BR-signaling mechanisms, leading to potential new targets for the genetic improvement of rice.

Functional analysis of BnMAR element in transgenic tobacco plants.

Scaffold/matrix attachment regions (S/MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin loops. Previous reports suggest that S/MARs elements may increase and stabilize the expression of transgene. In this study, DNA sequence with MAR characteristics has been isolated from B. napus . The BnMARs sequence was used to flank the CaMV35S-GUS-NOS expression cassette within the T-DNA of the plant expression vector pPZP212. These constructs were introduced into tobacco plants, respectively and the GUS reporter gene expression was investigated in stably transformed plants. When the forward BnMARs sequence was inserted into the upstream of CaMV35S promoter, the average GUS activities were much higher than those without BnMARs in transgenic tobacco. The GUS expression of M(+)35S:GUS, M(+)35S:GUSM(+) and M(+)35S:GUSM(-) constructs increased average 1.0-fold, with or without BnMARs located downstream of NOS. The GUS expression would not be affected when reverse BnMARs sequence inserted whether upstream of CaMV35S promoter or downstream of NOS. The GUS expression was affected a little when reverse BnMARs sequence was inserted the downstream of NOS and BnMARs could not act by serving as of promoter. The results showed that the presence of forward BnMARs sequence does have an obvious impact on enhancing downstream gene expression and its effect is unidirectional.

ZmCBF3 overexpression improves tolerance to abiotic stress in transgenic rice (Oryza sativa) without yield penalty.

Plant productivity is greatly affected by environmental stresses such as drought, salt, and freezing. We previously described a C-repeat binding transcription factor from maize (ZmCBF3) that was upregulated by both abscisic acid and low-temperature and actively expressed during embryogenesis. To understand the stress response in rice, transgenic ZmCBF3 rice with ubiquitin promoter was developed. T3 generation was planted and analyzed. The results showed that overexpression of ZmCBF3 in rice did not cause growth retardation under normal growth conditions with improved tolerance to drought, high-salt, and low-temperature stresses. Moreover, the transgenic rice grain yield was similar to wild type plants under normal conditions. The transgenic plants showed enhanced survival rate and reduced malondialdehyde content and relative conductivity under drought, salt, and low-temperature stresses. ZmCBF3 overexpression in transgenic rice increased the transcript levels of stress-induced genes and enhanced the tolerance to drought, salt, and low-temperature stresses.

HS1 Is Involved in Hygromycin Resistance Through Facilitating Hygromycin Phosphotransferase Transportation From Cytosol to Chloroplast.

The transportation of proteins encoded by nuclear genes from plant cytosol to chloroplast is essential for chloroplast functions. Proteins that have a chloroplast transit peptide (cTP) are imported into chloroplasts via translocases on the outer and inner chloroplast envelope. How proteins lacking transit sequence are imported into chloroplast remains largely unknown. During screening of an Arabidopsis population transformed with a hairpin RNA gene-silencing library, we identified some transgenic plants that had active expression of the selectable marker gene, hygromycin phosphotransferase (HPT), but were sensitive to the selection agent, hygromycin B (HyB). Mutant and complementation analysis showed that this HyB sensitivity of transgenic plants was due to silencing of the HS1 (Hygromycin-Sensitive 1) gene. HS1 is localized in the chloroplast and interacts physically with HPT in yeast cells and in planta. Fluorescence and immunoblotting analysis showed that HPT could not be transported effectively into chloroplasts in Aths1, which resulted in Aths1 is sensitivity to hygromycin on higher HyB-containing medium. These data revealed that HS1 is involved in HyB resistance in transgenic Arabidopsis through facilitating cytosol-chloroplast transportation of HPT. Our findings provide novel insights on transportation of chloroplast cTP-less proteins.

RNA-Seq Transcriptome Analysis of Rice Primary Roots Reveals the Role of Flavonoids in Regulating the Rice Primary Root Growth.

Flavonoids play important roles in root development and in its tropic responses, whereas the flavonoids-mediated changes of the global transcription levels during root growth remain unclear. Here, the global transcription changes in quercetin-treated rice primary roots were analyzed. Quercetin treatment significantly induced the inhibition of root growth and the reduction of H2O2 and O2- levels. In addition, the RNA-seq analysis revealed that there are 1243 differentially expressed genes (DEGs) identified in quercetin-treated roots, including 1032 up-regulated and 211 down-regulated genes. A gene ontology (GO) enrichment analysis showed that the enriched GO terms are mainly associated with the cell wall organization, response to oxidative stress, and response to hormone stimulus. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway analysis showed that the enriched DEGs are involved in phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. Moreover, the quercetin treatment led to an increase of the antioxidant enzyme activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in rice roots. Also, the quercetin treatment altered the DR5:GUS expression pattern in the root tips. All of these data indicated that the flavonoids-mediated transcription changes of genes are related to the genes involved in cell wall remodeling, redox homeostasis, and auxin signaling, leading to a reduced cell division in the meristem zone and cell elongation in the elongation zone of roots.

Effect of water-deficit on tassel development in maize.

Maize often exhibits asynchronous pollination under abiotic and biotic stress conditions; however, the molecular basis of this developmental deficiency has not been elucidated. Tassel development is a key process affecting the anthesis-silking interval (ASI) in maize. In this study, we showed that pollen shedding was delayed and ASI was significantly increased in B73 and Chang7-2 inbred lines under water deficit conditions, which resulted in longer barren tip length and decreased yields under both controlled and field conditions. Comparative transcriptome analysis performed on immature tassels derived from plants grown under well-watered and water deficit conditions identified 1931 and 1713 differentially expressed genes (DEGs) in B73 and Chang7-2, respectively. Further, 28 differentially co-expressed transcription factors were identified across both lines. Collectively, we demonstrated that the molecular regulation of tassel development is associated with water deficit stress at early vegetative stage in maize. This finding extends our understanding of the molecular basis of maize tassel development during abiotic stress.

Transcription factors NF-YA2 and NF-YA10 regulate leaf growth via auxin signaling in Arabidopsis.

In plants, leaf is crucial for photosynthesis and respiration. Leaf area and quantity are important for leaf vegetables to increase biomass. The process of leaf development involves coordinated regulation among small RNAs, transcription factors and hormones. Here, we found leaf size were regulated by transcription factors NF-YA2 and NF-YA10 in Arabidopsis. NF-YA2 and NF-YA10 overexpression increased biomass accumulation through promoting leaf growth and cell expansion. NF-YA2 and NF-YA10 were expressed in SAM and leaf vasculature. Endogenous IAA content reduced by 20% and 24% in transgenic Arabidopsis plants overexpressing NF-YA2 and NF-YA10 compared to wild-type plants. Chromatin immunoprecipitation assays revealed that NF-YA2 and NF-YA10 bound directly to the cis-element CCAAT in the promoter of the YUC2, and decreased the expression of YUC2, a YUCCA family gene. The auxin transporter gene PIN1 and auxin response factor1 and 2 (ARF1 and ARF2) genes, transcriptional repressors, were downregulated. These findings showed leaf development was regulated by NF-YA2 and NF-YA10 through the auxin-signaling pathway and may provide a new insight into the genetic engineering of vegetables biomass and crop productivity.