NKD Transcription Factors Are Central Regulators of Maize Endosperm Development

NAKED ENDOSPERM1 (NKD1) and NKD2 are duplicate INDETERMINATE DOMAIN (IDD) transcription factors important for maize (Zea mays) endosperm development. RNA-seq analysis of the nkd1 nkd2 mutant endosperm revealed that NKD1 and NKD2 influence 6.4% of the transcriptome in developing aleurone and 6.7% in starchy endosperm. Processes regulated by NKD1 and NKD2 include gene expression, epigenetic functions, cell growth and division, hormone pathways, and resource reserve deposition. The NKD1 and NKD2 proteins bind a consensus DNA sequence of TTGTCGT with slightly different properties. This motif was enriched in the promoters of gene transcripts differentially expressed (DE) in mutant endosperm. DE genes with a NKD binding motif in the 5′ promoter region were considered as likely direct targets of NKD1 and NKD2 regulation, and these putative direct target genes were notably enriched for storage proteins. Transcription assays demonstrate that NKD1 and NKD2 can directly regulate gene transcription, including activation of opaque2 and viviparous1 promoters. NKD2 functions as a negative regulator of nkd1 transcription, consistent with previously reported feedback regulation. NKD1 and NKD2 can homo- and heterodimerize through their ID domains. These analyses implicate NKD1 and NKD2 as central regulators of gene expression in developing maize endosperm

RNA interference knockdown of BRASSINOSTEROID INSENSITIVE1 in maize reveals novel functions for brassinosteroid signaling in controlling plant architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbril complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbriI-RNAi transgenic lines have compromised BR signaling. zmbriI-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbriI-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbriI-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbriI-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling

High-throughput linkage analysis of Mutator insertion sites in maize

Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator (Mu) in maize (Zea mays) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu-active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu-containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%

Analysis of a signal transduction pathway involved in leaf epidermis differentiation.

The major objective of this study was to identify and analyze signal transduction factors that function with the CR4 receptor kinase. We pursued this analysis in Arabidopsis. Analysis of other members of the ACR4 related receptor (CRR) family produced biochemical evidence consistent with some of them functioning in ACR4 signal transduction. Yeast 2-hybrid identified six proteins that interact with the cytoplasmic domain of ACR4, representing putative downstream signal transduction components. The interactions for all 6 proteins were verified by in vitro pull down assays. Five of the interacting proteins were phosphorylated by ACR4. We also identified candidate interactors with the extracellular TNFR domain. We hypothesize this may be the ligand binding domain for ACR4. In one approach, yeast 2-hybrid was again used and five candidate proteins identified. Nine additional candidates were identified in a genome wide scan of Arabidopsis amino acid sequences that threaded onto the TNF structure

The 46th Annual Maize Genetics Conference. Unlocking the Secrets of the Maize Genome

For the first time in its history, the Annual Maize Genetics Conference was held in Mexico City, near the center of origin for many Zea species, including maize. Maize research has made many contributions to our understanding of plant physiology and development, the regulation of transposable elements and chromosome structure, and the epigenetic control of gene expression. In addition to the reported advances in these research fields, this year's meeting emphasized the tremendous genetic diversity present within maize races. Explorations of this variation in studies of domestication, population genetics, and crop improvement hinted at the tremendous potential that lies within the maize genome. Tapping into this potential will soon be made easier as highlighted in a workshop outlining advances in maize genomics. Remarkable progress has been made toward sequencing the genic regions of the maize genome, and strategies to anchor and finish the entire maize gene space by 2006 were presented and discussed. This report highlights the new developments made in these areas of maize biology