Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in Arabidopsis thaliana via the THESEUS1 Pathway

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion

Genetic Analyses of Mechanisms of Compensation in Leaf Organogenesis

Leaf development relies on cell proliferation, post-mitotic cell expansion and the coordination of these processes. In several mutants of Arabidopsis thaliana impaired in cell proliferation, such as angustifolia3 (an3), leaf cells at their maturity are larger than those in wide-type leaves. Similar phenotypes have been reported in several mutants or transgenic plants in Oryza sativa and Nicotiana tabacum. These reports have suggested that magnitude of cell expansion is affected by cell proliferation activity during leaf development. This phenomenon, compensation, suggests the presence of coordination between cell proliferation and post-mitotic cell expansion during leaf development. However, knowledge about the oompensation was superficial and the mechanism(s) underlying compensation was unclear. The aim of his Ph.D. thesis is to elucidate the genetic framework that mediates compensation. To this end, he focused on an3 as a model case of compensation, since an3 is one of the best characterized compensation-exhibiting mutants. Since compensation consists of two different events, namely, the reduction in Cell number and the increase in cell size, he hypothesized that compensation can be theoretically separated into two distinct processes based on developmental processes; the induction process, which involves the reduction of cell proliferation; and the response process, during which cell expansion is enhanced. The originality in his Ph.D. thesis is that he investigated separately these two processes: induction and response of compensation. He adopted a strategy to genetically dissect the compensation syndrome using knowledge on anatomy of leaf development. Namely, to address how aberrant cell enlargement is induced and how cell expansion system responds to the inductive condition during compensation, he planned to combine mutants which have a specific defect in each process. However, in many reports about dwarf mutants, histological analysis revealing number and size of cells within the leaves has been insufficient. Thus, the primary defect(s) contributing to changes in organ size in these dwarf mutants remained unclear. Therefore, he analyzed the cell number and size within leaves in several known dwarf mutants. As a result, unexpectedly, most of the well-known dwarf mutants had detects in both cell proliferation and cell expansion and they were wrongly reported from very limited, fragmental observations. Thus, he found that it is necessary to newly isolate cell-number or -size specific mutants to analyze the induction and response processes of compensation. From a large collection of leaf-size mutants established in the laboratory he belongs, he successfully isolated several mutant lines with a spectfic defect in leaf cell number or size. Then, he utilized these mutants to analyze the induction or response process of compensation. In the analysis of "induction" process of compensation, he selected six lines of utants from the above-mentioned mutants isolated, named otigocellula (oli), which had a specific decrease in cell number. Although the induction of compensation is thought to be closely linked to a decrease in cell number, oli mutants did not exhibit compensation. Thus, he thought that the induction of compensation requires not only a decrease in cell number but also some special conditions. But before this study, nothing was known for the condition required for induction of compensation. To clarify conditions, he produced double mutants among oli mutants in which the extent of decrease in cell number is variable. As a result, when the leaf cell number in oli double mutant was significantly decreased than each parental single oli mutant, the leaf cell size was strongly increased. This result indicates a possibillity that the induction of oompensation depends on a decrease in cell number in a threshold-dependent manner. To further address whether a further decrease in cell number affects cetl size, next, he constructed double mutants between the an3 and the oli mutants. As a result, most of oli an3 double mutants had much fewer and significantly Darger leaf cells than the an3. Exceptionally, oli1 an3 double mutant had a similar number and size of leaf cells to an3. These results indicated that the further decrease in cell number induces an additional and proportional cell enlargement, when cell number fall below a threshold level. It also revealed that the OLI1 and AN3 genes have a function in cell proliferation in the same genetic pathway. Some researchers have regarded compensation as a result of uncoupling of cell growth and cell division from cell growth. If compensation results from an uncoupling of celf growth and cell division, cell size should be larger in proportion to any decreases in cell number. However, identification of threshold ruled out this possibillty. These findings demonstrate that compensation is not a mere uncoupling of cell cycling and cell enlargement and suggest the existence of yet unknown genetic network between cell proliferation and cell expansion in leaf organogenesis. For the analysis of "responsei" process of compensation, he isolated and utilized ten lines of mutant, that he named extra-small sisters (xs), which have a specific defect in cell expansion but have normal cell numbers in leaves. In the xs single mutants, the palisade cell sizes in mature leaves are about 20 to 50% smaller than wild-type cells. Then, he produced double mutants between xs mutants and an3 to genetically dissect the cell expansion system(s) bunderiying compensation in the an3 mutant. As a result, phenotypes of the palisade cell sizes in all combinations of xs an3 double mutants fall into three classes. In the first class, the compensation was significantly suppressed. Conversely, in the second class, the defective cell expansion conferred by the xs mutations was significantly suppressed by the an3 mutation, and the cell sizes were close to that in the an3 single mutant. The residuafi xs mutations had effects additive to those of the an3 mutation on cell expansion. The endopolyploidy levels in the first class of mutants were decreased, unaffected, or increased, as compared to those in wild type, suggesting that the abnormally enhanced cell expansion observed in an3 is mediated, at least in part, by ploidy-independent mechanisms. Altogether, these results suggest that a defect in cell proliferation in leaf primordia enhances a part of the cell expansion system required for normal cell expansion. Taken all together, the present study has revealed important clues of leaf size control: the induction process of compensation deponds on a decrease in cete number in a threshold-dependent manner; the response process of oompensation depends on a specific enhancement of normal processes of cell expansion control; and component of genetic networks for the control of leaf cell proliferation and celt expansion

Herbicide tolerance-assisted multiplex targeted nucleotide substitution in rice

Acetolactate synthase (ALS) catalyzes the initial step in the biosynthesis of branched-chain amino acids, and is highly conserved from bacteria to higher plants. ALS is encoded by a single copy gene in rice genome and is a target enzyme of several classes of herbicides. Although ALS mutations conferring herbicide-resistance property to plants are well documented, effect of Imazamox (IMZ) on rice and the mutations in ALS correlated with IMZ tolerance were unclear. In this article, the effect of IMZ on rice calli and seedlings in tissue culture conditions were evaluated. Also, the ALS A96V mutation was confirmed to improve IMZ tolerance of rice calli. Based on these results, ALS-assisted multiplex targeted base editing in rice was demonstrated in combination with Target-AID, a CRISPR/Cas9-cytidine deaminase fusion system [1], [2]

Correction: Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling.

[This corrects the article DOI: 10.1371/journal.pgen.1008873.]

Suppression of class I compensated cell enlargement by xs2 mutation is mediated by salicylic acid signaling

The regulation of leaf size has been studied for decades. Enhancement of post-mitotic cell expansion triggered by impaired cell proliferation in Arabidopsis is an important process for leaf size regulation, and is known as compensation. This suggests a key interaction between cell proliferation and cell expansion during leaf development. Several studies have highlighted the impact of this integration mechanism on leaf size determination; however, the molecular basis of compensation remains largely unknown. Previously, we identified extra-small sisters (xs) mutants which can suppress compensated cell enlargement (CCE) via a specific defect in cell expansion within the compensation-exhibiting mutant, angustifolia3 (an3). Here we revealed that one of the xs mutants, namely xs2, can suppress CCE not only in an3 but also in other compensation-exhibiting mutants erecta (er) and fugu2. Molecular cloning of XS2 identified a deleterious mutation in CATION CALCIUM EXCHANGER 4 (CCX4). Phytohormone measurement and expression analysis revealed that xs2 shows hyper activation of the salicylic acid (SA) response pathway, where activation of SA response can suppress CCE in compensation mutants. All together, these results highlight the regulatory connection which coordinates compensation and SA response

GROWTH-REGULATING FACTOR 9 negatively regulates arabidopsis leaf growth by controlling <i>ORG3</i> and restricting cell proliferation in leaf primordia

<div><p>Leaf growth is a complex process that involves the action of diverse transcription factors (TFs) and their downstream gene regulatory networks. In this study, we focus on the functional characterization of the <i>Arabidopsis thaliana</i> TF GROWTH-REGULATING FACTOR9 (GRF9) and demonstrate that it exerts its negative effect on leaf growth by activating expression of the bZIP TF <i>OBP3-RESPONSIVE GENE 3</i> (<i>ORG3</i>). While <i>grf9</i> knockout mutants produce bigger incipient leaf primordia at the shoot apex, rosette leaves and petals than the wild type, the sizes of those organs are reduced in plants overexpressing <i>GRF9</i> (<i>GRF9ox</i>). Cell measurements demonstrate that changes in leaf size result from alterations in cell numbers rather than cell sizes. Kinematic analysis and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay revealed that <i>GRF9</i> restricts cell proliferation in the early developing leaf. Performing <i>in vitro</i> binding site selection, we identified the 6-base motif 5'-CTGACA-3' as the core binding site of GRF9. By global transcriptome profiling, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) we identified <i>ORG3</i> as a direct downstream, and positively regulated target of GRF9. Genetic analysis of <i>grf9 org3</i> and <i>GRF9ox org3</i> double mutants reveals that both transcription factors act in a regulatory cascade to control the final leaf dimensions by restricting cell number in the developing leaf.</p></div