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

Higher Plant Cytochrome b5 Polypeptides Modulate Fatty Acid Desaturation

BACKGROUND: Synthesis of polyunsaturated fatty acids (PUFAs) in the endoplasmic reticulum of plants typically involves the fatty acid desaturases FAD2 and FAD3, which use cytochrome b(5) (Cb5) as an electron donor. Higher plants are reported to have multiple isoforms of Cb5, in contrast to a single Cb5 in mammals and yeast. Despite the wealth of information available on the roles of FAD2 and FAD3 in PUFA synthesis, information regarding the contributions of various Cb5 isoforms in desaturase-mediated reactions is limited. RESULTS: The present functional characterization of Cb5 polypeptides revealed that all Arabidopsis Cb5 isoforms are not similarly efficient in ω-6 desaturation, as evidenced by significant variation in their product outcomes in yeast-based functional assays. On the other hand, characterization of Cb5 polypeptides of soybean (Glycine max) suggested that similar ω-6 desaturation efficiencies were shared by various isoforms. With regard to ω-3 desaturation, certain Cb5 genes of both Arabidopsis and soybean were shown to facilitate the accumulation of more desaturation products than others when co-expressed with their native FAD3. Additionally, similar trends of differential desaturation product accumulation were also observed with most Cb5 genes of both soybean and Arabidopsis even if co-expressed with non-native FAD3. CONCLUSIONS: The present study reports the first description of the differential nature of the Cb5 genes of higher plants in fatty acid desaturation and further suggests that ω-3/ω-6 desaturation product outcome is determined by the nature of both the Cb5 isoform and the fatty acid desaturases

Expression profiling of squalene monooxygenase genes of sterol biosynthesis path [abstract]

Abstract only availableFaculty Mentor: Henry T. Nguyen, AgronomyPhytosterol are a group of sterol alcohols and these phytochemicals occur naturally in plants. They are important as structure components of plasma membranes and precursor of steroidal hormones in plants. The sterol biosynthetic pathway has been a topic of intense researches in recent years and several genes have been identified and functionally characterized in the model plant Arabidopsis thaliana. In this project, we are investigating the differences in the gene expression level of squalene monooxygenase(SQM) gene family members, catalyzing the first step toward sterol biosynthesis in the post squalene pathway in Arabidopsis thaliana. Samples of germinating seed and vegetative tissues were collected and gene expression studies with real - time reverse transcription polymerase chain reaction(RT-PCR) approach was performed. The SQM members showed differential expression during germination, seedling establishment and early vegetative stages. All the SQM gene, except SQM4, were found to be up-regulated in the imbibition stage following stratification treatment. SQM4 was absent during radicle emergence

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

WOS: 000360930600062PubMed ID: 26162429Brassinosteroids (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.Iowa State University Plant Sciences Institute; Genetics, Development, and Cell Biology Department; Ministry of National Education, Republic of TurkeyMinistry of National Education - TurkeyThis work was supported by the Iowa State University Plant Sciences Institute and the Genetics, Development, and Cell Biology Department and by the Ministry of National Education, Republic of Turkey (education fellowship to G.K.)

The 16∶2 and 18∶2 content of yeast co-expressing <i>Cb5</i> and <i>FAD2</i> of soybean.

<p>Cultures were induced with galactose and allowed to grow 28°C for 48 h (A) or at 15°C for 96 h (B). FAMES were analyzed by GC-FID. Values are expressed as molar percentage of total FAs and represent average and SD of three independent cultures. W = wild type yeast (empty vector pESC control); M = mutant <i>cb5</i> yeast. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031370#pone.0031370.s003" target="_blank">Table S2</a> for detailed fatty acid composition.</p

The 18∶3 content of yeast co-expressing <i>Cb5</i> and <i>FAD3</i> of <i>Arabidopsis</i>.

<p>Conditions same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031370#pone-0031370-g005" target="_blank">Figure 5</a>. W = wild type yeast (empty vector pESC control); M = mutant <i>cb5</i> yeast. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031370#pone.0031370.s004" target="_blank">Table S3</a> for detailed fatty acid composition.</p

Cytochrome <i>b</i>5 (<i>Cb5</i>) genes of soybean.

<p>Cytochrome <i>b</i>5 (<i>Cb5</i>) genes of soybean.</p