Constructing and testing a genetic network for controlling seed germination in Arabidopsis

The funiculus (FUN) is continuous with the chalazal seed coat (CSC) during seed development. We previously reported that AUXIN SIGNALING F-BOX PROTEIN 1 and 5 (AFB1 and 5) expressed in FUN and CSC suppress seed germination. To find the genes regulated by AFBs in the seed germination process, we first determined the expression patterns of AFBs in the FUN-CSC continuum based on the publicly available data. We found that TIR1, AFB1, and AFB4 (the AFB1 group) exhibit a down-expression gradient and AFB2, AFB3, and AFB5 (the AFB5 group) an up-expression gradient from FUN to CSC. The estimated mRNA concentrations of the three AFBs in each group are highly linearly correlated in FUN, CSC, and the distal (away from CSC) seed coat (DSC) region. We then searched for auxin-regulated genes that exhibited a down-expression or an up- expression gradient from FUN to CSC. We found 118 such genes that were assigned into four groups based on their response mode (positive or negative) to auxin and expression gradient direction (up or down) from FUN to CSC: 1) downregulated presumably by the AFB1 group with an up-expression gradient, 2) upregulated presumably by the AFB5 group with an up-expression gradient, 3) upregulated presumably by the AFB1 group with a down-expression gradient, and 4) downregulated presumably by the AFB5 group with a down-expression gradient. These four groups were further broken down into 12 subgroups based on linear correlation analysis of their mRNA concentrations in FUN, CSC, and DSC. Three of the 12 subgroups, including a total of 30 genes, were investigated further because 21 of the 30 genes are known or highly likely to function in the seed germination process. We have tested seed germination in the mutants of five of the nine genes (two alleles/gene) unknown for their involvement in seed germination, with the mutants of three genes exhibiting delayed germination and the mutants of the other two genes exhibiting hastened germination. The experimental results support the validity of the approach used to predict the involvement of these genes in seed germination. Based on the publicly available data and data from this investigation, we constructed a genetic network that consists of the 30 identified proteins, the six AFBs, BES1 and BZR1 involved in brassinosteroid signaling, and ABI3, 4, and 5 involved in ABA signaling. This genetic network should provide a valuable framework and new clues for future studies of the molecular mechanism controlling seed germination.Plant Biology, Ecology and Evolutio

Predicting maternal auxin-signaling networks for seed dormancy regulation in Arabidopsis

Auxin, like ABA, promotes seed dormancy, but how auxin promotes seed dormancy is not well understood. In studying seed dormancy regulation, we found that AUXIN SIGNALING F-BOX PROTEIN 1 (AFB1) and 5 maternally promoted seed dormancy and AFB1 had a stronger effect than AFB5. AFB1 and 5 were expressed in the funiculus and the chalazal seed coat at the mature embryo stage, and AFB1, not AFB5, was also transiently expressed in a small chalazal seed coat region surrounding the remnant funiculus during seed imbibition. Analysis of publically available datasets for genes expressed in the funiculus and seed coat at the mature embryo stage allowed the assignment of the six AFBs into two groups: TIR1, AFB1 and 4 as the first group with higher expression levels in the funiculus than in the chalazal seed coat, and AFB2, 3 and 5 as the second group with higher expression levels in the chalazal seed coat than in the funiculus. It was then assumed that auxin-upregulated and -downregulated genes associated with the first AFB group should be expressed at higher and lower levels in the funiculus than in the chalazal seed coat, respectively, and the reverse is assumed for those associated with the second AFB group. Three potential auxin-signaling networks including 30 genes were identified based on these assumptions and high linear correlation in expression within each group. These networks do not overlap in components and two-thirds of the genes are known or predicted to function in seed germination either positively or negatively. The presence of both positive and negative regulators in each of the networks is consistent with the plant’s ability to either remain dormant or go into germination in response to environmental conditions. The identified components of the networks also suggest interactions of auxin with other hormones in seed dormancy regulation.Plant Biology, Ecology and Evolutio

AFB1 and 5 act maternally outside the seed to promote seed growth in Arabidopsis

The plant hormone auxin in maternal tissues plays a major role in promoting seed growth. However, whether auxin affects seed growth in maternal tissues in or outside the seed is unclear. In the process of investigating the roles of the SKP1-CULLIN-F- BOX (SCF) ubiquitin ligases in Arabidopsis reproductive development, using protein extracts from inflorescence tissues, we found that, among the six auxin receptors, only AUXIN SIGNALING F-BOX1 (AFB1) and 5 (AFB5) were consistently co- immunoprecipitated with the ARABIDOPSIS SKP1-LIKE1 protein. This result suggests that AFB1 and 5 play major roles in reproductive development. We then found that seeds from afb1 and 5 mutants exhibited reduced sizes (weights). The largest reduction in seed weight occurred in afb5-6; the average weight/1000 dry seeds of afb5-6 was approximately 76% of that of the wild type. The seed weights were further investigated in AFB1 and AFB5 transgenic plants with the transgenes driven by their respective promoters or the ASK1 promoter. The results from the T₂ seeds and homozygous T₃ and T₄ seeds suggest that the wild-type levels of the two transcripts are not limiting for seed growth and farther increasing their levels adversely affects seed growth. Histochemical studies of transgenic plants harboring the AFB1:GUS or AFB5:GUS transgene revealed that AFB1 and AFB5 are not expressed in the seed but in the vascular tissue in the fruit wall and the funiculus. Taken together, these results show that AFB1 and AFB5 promote seed growth in maternal tissues outside the seed.Plant Biology, Ecology and Evolutio

Loss-of-function mutants and overexpression lines of the Arabidopsis cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS exhibit different defects in prophase-I meiocytes but produce the same abnormal meiotic products

In Arabidopsis, loss-of-function mutations in the A-type cyclin CYCA1;2/TARDY ASYNCHRONOUS MEIOSIS (TAM) gene lead to the production of abnormal meiotic products including triads and dyads. Here we report that overexpression of TAM by the ASK1:TAM transgene also led to the production of triads and dyads in meiosis, as well as shriveled seeds, in a dominant fashion. However, the partial loss-of-function mutant tam-1, an ASK1:TAM line, and the wild type differed in dynamic changes in chromosome thread thickness from zygotene to diplotene. We also found that the pericentromeric heterochromatin regions in male meiocytes in tam-1 and tam-2 (a null allele) frequently formed a tight cluster at the pachytene and diplotene stages, in contrast to the infrequent occurrences of such clusters in the wild type and the ASK1:TAM line. Immunolocalization studies of the chromosome axial component ASY1 revealed that ASY1 was highly expressed at the appropriate male meiotic stages but not localized to the chromosomes in tam-2. The level of ASY1, however, was greatly reduced in another ASK1:TAM line with much overexpressed TAM. Our results indicate that the reduction and increase in the activity of TAM differentially affect chromosomal morphology and the action of ASY1 in prophase I. Based on these results, we propose that either the different meiotic defects or a common defect such as missing ASY1 on the chromosomal axes triggers a hitherto uncharacterized cell cycle checkpoint in the male meiocytes in the tam mutants and ASK1:TAM lines, leading to the production of the same abnormal meiotic products.Plant Biology, Ecology and Evolutio

A new gene for regulation of epidermal cell production in Arabidopsis cotyledons

Plant Biology, Ecology and Evolutio

Effect of frequency coupling on stability analysis of a grid-connected modular multilevel converter system

Due to the internal dynamics of the modular multilevel converter (MMC), the coupling between the positive and negative sequences in impedance, which is defined as frequency coupling, inherently exists in MMC. Ignoring the frequency coupling of the MMC impedance model may lead to inaccurate stability assessment, and thus the multi-input multi-output (MIMO) impedance model has been developed to consider the frequency coupling effect. However, the generalized Nyquist criterion (GNC), which is used for the stability analysis of an MIMO model, is more complicated than the stability analysis method applied on single-input-single-output (SISO) models. Meanwhile, it is not always the case that the SISO model fails in the stability assessment. Therefore, the conditions when the MIMO impedance model needs to be considered in the stability analysis of an MMC system should be analyzed. This paper quantitatively analyzes the effect of frequency coupling on the stability analysis of grid-connected MMC, and clarifies the frequency range and grid conditions that the coupling effect required to be considered in the stability analysis. Based on the quantitative relations between the frequency coupling and the stability analysis of the grid-connected MMC system, a simple and accurate stability analysis method for the grid-connected MMC system is proposed, where the MIMO impedance model is applied when the frequency coupling has a significant effect and the SISO impedance model is used if the frequency coupling is insignificant