Transcriptional regulation of nitrogen-associated metabolism and growth.

Nitrogen is an essential macronutrient for plant growth and basic metabolic processes. The application of nitrogen-containing fertilizer increases yield, which has been a substantial factor in the green revolution1. Ecologically, however, excessive application of fertilizer has disastrous effects such as eutrophication2. A better understanding of how plants regulate nitrogen metabolism is critical to increase plant yield and reduce fertilizer overuse. Here we present a transcriptional regulatory network and twenty-one transcription factors that regulate the architecture of root and shoot systems in response to changes in nitrogen availability. Genetic perturbation of a subset of these transcription factors revealed coordinate transcriptional regulation of enzymes involved in nitrogen metabolism. Transcriptional regulators in the network are transcriptionally modified by feedback via genetic perturbation of nitrogen metabolism. The network, genes and gene-regulatory modules identified here will prove critical to increasing agricultural productivity.

Maize BIG GRAIN1 homolog overexpression increases maize grain yield.

The Zea Mays BIG GRAIN 1 HOMOLOG 1 (ZM-BG1H1) was ectopically expressed in maize. Elite commercial hybrid germplasm was yield tested in diverse field environment locations representing commercial models. Yield was measured in 101 tests across all 4 events, 26 locations over 2 years, for an average yield gain of 355 kg/ha (5.65 bu/ac) above control, with 83% tests broadly showing yield gains (range +2272 kg/ha to -1240 kg/ha), with seven tests gaining more than one metric ton per hectare. Plant and ear height were slightly elevated, and ear and tassel flowering time were delayed one day, but ASI was unchanged, and these traits did not correlate to yield gain. ZM-BG1H1 overexpression is associated with increased ear kernel row number and total ear kernel number and mass, but individual kernels trended slightly smaller and less dense. The ZM-BG1H1 protein is detected in the plasma membrane like rice OS-BG1. Five predominant native ZM-BG1H1 alleles exhibit little structural and expression variation compared to the large increased expression conferred by these ectopic alleles.

Protoplast Isolation and Transfection in Maize.

Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the isolation of large quantities of healthy protoplasts from the tissue of interest. Herein, we describe protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. The isolation protocol yields approximately 1.8-1.9 × 107 protoplasts with 80-90% viability from 6 g of etiolated leaf tissue, and the polyethylene glycol-mediated transfection protocol results in 55-58% transfection efficiency. The transfection protocol describes the use of a dual-expression vector that carries the coding sequence for two fluorescent proteins (FPs), one driven by a constitutive promoter for normalization for transfection efficiency and the other driven by the construct of interest. The use of a dual-FP expression vector eliminates the need for co-transfection and separate steps for enzymatic/substrate processing as required for luciferase-based assays. These protocols have been tested on leaf tissue from the maize genotypes B73 and PHR03 and, as written, can be completed in 24 h.

A small, novel protein highly conserved in plants and animals promotes the polarized growth and division of maize leaf epidermal cells.

Plant cell shapes are defined by their surrounding walls, but microtubules and F-actin both play critical roles in cell morphogenesis by guiding the deposition of wall materials in expanding cells. Leaf epidermal cells have lobed shapes, which are thought to arise through a microtubule-dependent pattern of locally polarized growth. We have isolated a recessive mutation, brk1, which blocks the formation of epidermal cell lobes in the maize leaf. Mutant epidermal cells expand to the same extent as wild-type cells but fail to establish polar growth sites from which lobes arise. In expanding brk1 epidermal cells, microtubule organization differs little from that in wild-type, but localized enrichments of cortical F-actin seen at the tips of emerging lobes in wild-type cells fail to form. These observations suggest a critical role for F-actin in lobe formation and together with additional effects of brk1 on the morphogenesis of stomata and hairs suggest that Brk1 promotes multiple, actin-dependent cell polarization events in the developing leaf epidermis. The Brk1 gene encodes a novel, 8 kD protein that is highly conserved in plants and animals, suggesting that BRK1-related proteins may function in actin-dependent aspects of cell polarization in a wide spectrum of eukaryotic organisms.

BRICK1/HSPC300 functions with SCAR and the ARP2/3 complex to regulate epidermal cell shape in Arabidopsis.

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, is essential for a variety of eukaryotic cellular processes, including epidermal cell morphogenesis in Arabidopsis thaliana. Efficient nucleation of actin filaments by the Arp2/3 complex requires the presence of an activator such as a member of the Scar/WAVE family. In mammalian cells, a multiprotein complex consisting of WAVE, PIR121/Sra-1, Nap1, Abi-2 and HSPC300 mediates responsiveness of WAVE to upstream regulators such as Rac. Essential roles in WAVE complex assembly or function have been demonstrated for PIR121/Sra-1, Nap1 and Abi-2, but the significance of HSPC300 in this complex is unclear. Plant homologs of all mammalian WAVE complex components have been identified, including HSPC300, the mammalian homolog of maize BRICK1 (BRK1). We show that, like mutations disrupting the Arabidopsis homologs of PIR121/Sra-1, Nap1 and Scar/WAVE, mutations in the Arabidopsis BRK1 gene result in trichome and pavement cell morphology defects (and associated alterations in the F-actin cytoskeleton of expanding cells) similar to those caused by mutations disrupting the ARP2/3 complex itself. Analysis of double mutants provides genetic evidence that BRK1 functions in a pathway with the ARP2/3 complex. BRK1 is required for accumulation of SCAR1 protein in vivo, potentially explaining the apparently essential role of BRK1 in ARP2/3 complex function.

Three Brick genes have distinct functions in a common pathway promoting polarized cell division and cell morphogenesis in the maize leaf epidermis.

We have taken a genetic approach to investigating cytoskeleton-dependent mechanisms governing cell morphogenesis in the maize leaf epidermis. Previously, we showed that the Brick1 (Brk1) gene is required for the formation of epidermal cell lobes as well as for properly polarized divisions of stomatal subsidiary mother cells, and encodes an 8 kDa protein highly conserved in plants and animals. Here, we show that two additional Brick genes, Brk2 and Brk3, are involved in the same aspects of epidermal cell morphogenesis and division. As shown previously for Brk1, analysis of the cytoskeleton shows that Brk2 and Brk3 are required for the formation of local F-actin enrichments associated with lobe outgrowth in wild-type cells. Analysis of brk1;brk2, brk1;brk3 and brk2;brk3 double mutants shows that their phenotypes are the same as those of brk single mutants. Mosaic analysis shows that Brk1 acts non cell-autonomously over a short distance. By contrast, Brk2 and Brk3 act cell-autonomously to promote pavement cell lobe formation, but Brk3 acts non cell-autonomously, and Brk2 partially non cell-autonomously, to promote polarized subsidiary mother cell divisions. Together, these observations indicate that all three Brk genes act in a common pathway in which each Brk gene has a distinct function. Recent work demonstrating a function for the mammalian homolog of BRK1 (HSPC300) in activation of Arp2/3-dependent actin polymerization implicates the Brk pathway in local regulation of actin polymerization in plant cells.