The TMK Subfamily of Receptor-Like Kinases in Arabidopsis Display an Essential Role in Growth and a Reduced Sensitivity to Auxin

Mechanisms that govern the size of plant organs are not well understood but believed to involve both sensing and signaling at the cellular level. We have isolated loss-of-function mutations in the four genes comprising the transmembrane kinase TMK subfamily of receptor-like kinases (RLKs) in Arabidopsis. These TMKs have an extracellular leucine-rich-repeat motif, a single transmembrane region, and a cytoplasmic kinase domain. While single mutants do not display discernable phenotypes, unique double and triple mutant combinations result in a severe reduction in organ size and a substantial retardation in growth. The quadruple mutant displays even greater severity of all phenotypes and is infertile. The kinematic studies of root, hypocotyl, and stamen filament growth reveal that the TMKs specifically control cell expansion. In leaves, TMKs control both cell expansion and cell proliferation. In addition, in the tmk double mutants, roots and hypocotyls show reduced sensitivity to applied auxin, lateral root induction and activation of the auxin response reporter DR5: GUS. Thus, taken together with the structural and biochemical evidence, TMKs appear to orchestrate plant growth by regulation of both cell expansion and cell proliferation, and as a component of auxin signaling.     

Indole-3-glycerol phosphate, a branchpoint of indole-3-acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana

The phytohormone indole-3-acetic acid (IAA) plays a vital role in plant growth and development as a regulator of numerous biological processes. Its biosynthetic pathways have been studied for decades. Recent genetic and in vitro labeling evidence indicates that IAA in Arabidopsis thaliana and other plants is primarily synthesized from a precursor that is an intermediate in the tryptophan (Trp) biosynthetic pathway. To determine which intermediate(s) acts as the possible branchpoint for the Trp-independent IAA biosynthesis in plants, we took an in vivo approach by generating antisense indole-3-glycerol phosphate synthase (IGS) RNA transgenic plants and using available Arabidopsis Trp biosynthetic pathway mutants trp2-1 and trp3-1. Antisense transgenic plants display some auxin deficient-like phenotypes including small rosettes and reduced fertility. Protein gel blot analysis indicated that IGS expression was greatly reduced in the antisense lines. Quantitative analyses of IAA and Trp content in antisense IGS transgenic plants and Trp biosynthetic mutants revealed striking differences. Compared with wild-type plants, the Trp content in all the transgenic and mutant plants decreased significantly. However, total IAA levels were significantly decreased in antisense IGS transgenic plants, but remarkably increased in trp3-1 and trp2-1 plants. These results suggest that indole-3-glycerol phosphate (IGP) in the Arabidopsis Trp biosynthetic pathway serves as a branchpoint compound in the Trp-independent IAA de novo biosynthetic pathway.     

Tryptophan auxotroph mutants suppress the superroot2 phenotypes,modulating IAA biosynthesis in arabidopsis

Plant phytohormone, Indole-3-acetic acid (IAA ), is synthesized by tryptophan (trp) dependent and independent pathway. Here we report that tryptophan auxotroph mutants completely suppressed the abnormalities of auxin over production mutant, superroot2. SUR2 is considered to modulate Trp dependent pathway, resulting IAA accumulation in Arabidopsis. Tryptophan auxotroph mutants showed hyper-sensitivity to the auxin polar transport inhibitor, NPA, on the phenotype of reduced gravitropism. These results together with the results of histochemical analyses, tryptophan auxotroph mutants seem to have a complete defect in Trp dependent IAA biosynthesis pathway, and it is also suggested that the Trp dependent pathway is responsible for the normal root gravitropism.Key words: Auxin, Trp dependent pathway, trp mutants, sur2, arabidopsis     

The BIG gene is required for auxin-mediated organ growth in Arabidopsis

Control of organ size by cell expansion and cell proliferation is a fundamental process during development, but the importance of BIG in this process is still poorly understood. Here, we report the isolation and characterization of a new allele mutant of BIG in Arabidopsis: big-j588. The mutant displayed small aerial organs that were characterized by reduced cell size in the epidermis and short roots with decreased cell numbers. The big-j588 axr1 double and big-j588 arf7 arf19 triple mutants displayed more severe defects in leaf expansion and root elongation than their parents, implying BIG is involved in auxin-dependent organ growth. Genetic analysis suggests that BIG may act synergistically with PIN1 to affect leaf growth. The PIN1 protein level decreased in both the root cells and the tips of leaf pavement cell lobes of big-j588. Further analysis showed that the auxin maxima in the roots and the leaves of big-j588 decreased. Therefore, we concluded that the small leaves and the short roots of big-j588 were associated with reduction of auxin maxima. Overall, our study suggested that BIG is required for Arabidopsis organ growth via auxin action.     

The Arabidopsis SMO2, a homologue of yeast TRM112, modulates progression of cell division during organ growth

Cell proliferation is integrated into developmental progression in multicellular organisms, including plants, and the regulation of cell division is of pivotal importance for plant growth and development. Here, we report the identification of an Arabidopsis SMALL ORGAN 2 (SMO2) gene that functions in regulation of the progression of cell division during organ growth. The smo2 knockout mutant displays reduced size of aerial organs and shortened roots, due to the decreased number of cells in these organs. Further analyses reveal that disruption of SMO2 does not alter the developmental timing but reduces the rate of cell production during leaf and root growth. Moreover, smo2 plants exhibit a constitutive activation of cell cycle‐related genes and over‐accumulation of cells expressing CYCB1;1:β‐glucuronidase (CYCB1;1:GUS) during organogenesis, suggesting that smo2 has a defect in G₂–M phase progression in the cell cycle. SMO2 encodes a functional homologue of yeast TRM112, a plurifunctional component involved in a few cellular events, including tRNA and protein methylation. In addition, the mutation of SMO2 does not appear to affect endoreduplication in Arabidopsis leaf cells. Taken together we postulate that Arabidopsis SMO2 is a conserved yeast TRM112 homologue and SMO2‐mediated cellular events are required for proper progression of cell division in plant growth and development.     

Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis

Auxin plays a key role in embryogenesis and seedling development, but the auxin sources for the two processes are not defined. Here, we demonstrate that auxin synthesized by the YUCCA (YUC) flavin monooxygenases is essential for the establishment of the basal body region during embryogenesis and the formation of embryonic and postembryonic organs. Both YUC1 and YUC4 are expressed in discrete groups of cells throughout embryogenesis, and their expression patterns overlap with those of YUC10 and YUC11 during embryogenesis. The quadruple mutants of yuc1 yuc4 yuc10 yuc11 fail to develop a hypocotyl and a root meristem, a phenotype similar to those of mp and tir1 afb1 afb2 afb3 auxin signaling mutants. We further show that YUC genes play an essential role in the formation of rosette leaves by analyzing combinations of yuc mutants and the polar auxin transport mutants pin1 and aux1. Disruption of YUC1, YUC4, or PIN1 alone does not abolish leaf formation, but the triple mutant yuc1 yuc4 pin1 fails to form leaves and flowers. Furthermore, disruption of auxin influx carrier AUX1 in the quadruple mutant yuc1 yuc2 yuc4 yuc6, but not in wild-type background, phenocopies yuc1 yuc4 pin1, demonstrating that auxin influx is required for plant leaf and flower development. Our data demonstrate that auxin synthesized by the YUC flavin monooxygenases is an essential auxin source for Arabidopsis thaliana embryogenesis and postembryonic organ formation.     

The Arabidopsis auxin-inducible gene ARGOS controls lateral organ size

During plant development, the final size of an organ is regulated and determined by various developmental signals; however, the molecular mechanisms by which these signals are transduced and the mediators involved are largely unknown. Here, we show that ARGOS, a novel Arabidopsis gene that is highly induced by auxin, is involved in organ size control. Transgenic plants expressing sense or antisense ARGOS cDNA display enlarged or reduced aerial organs, respectively. The alteration in organ size is attributable mainly to changes in cell number and the duration of organ growth. Ectopic expression of ARGOS prolongs the expression of AINTEGUMENTA (ANT) and CycD3;1 as well as the neoplastic activity of leaf cells. Moreover, organ enlargement in plants overexpressing ARGOS can be blocked by the loss of function of ANT, implying that ARGOS functions upstream of ANT to affect the meristematic competence of organ cells. The induction of ARGOS by auxin is attenuated or abolished in auxin-resistant1 (axr1), and overexpression of ARGOS partially restores axr1 organ development. These results suggest that ARGOS may transduce auxin signals downstream of AXR1 to regulate cell proliferation and organ growth through ANT during organogenesis.     

Gene expression profile of zeitlupe/lov kelch protein1 T-DNA insertion mutants in Arabidopsis thaliana: Downregulation of auxin-inducible genes in hypocotyls

Elongation of hypocotyl cells has been studied as a model for elucidating the contribution of cellular expansion to plant organ growth. ZEITLUPE (ZTL) or LOV KELCH PROTEIN1 (LKP1) is a positive regulator of warmth-induced hypocotyl elongation under white light in Arabidopsis, although the molecular mechanisms by which it promotes hypocotyl cell elongation remain unknown. Microarray analysis showed that 134 genes were upregulated and 204 genes including 15 auxin-inducible genes were downregulated in the seedlings of 2 ztl T-DNA insertion mutants grown under warm conditions with continuous white light. Application of a polar auxin transport inhibitor, an auxin antagonist or an auxin biosynthesis inhibitor inhibited hypocotyl elongation of control seedlings to the level observed with the ztl mutant. Our data suggest the involvement of auxin and auxin-inducible genes in ZTL-mediated hypocotyl elongation.     

Helical Growth of the Arabidopsis Mutant tortifolia2 Does Not Depend on Cell Division Patterns but Involves Handed Twisting of Isolated Cells

Several factors regulate plant organ growth polarity. tortifolia2 (tor2), a right-handed helical growth mutant, has a conservative replacement of Arg-2 with Lys in the α-tubulin 4 protein. Based on a published high-resolution (2.89 Å) tubulin structure, we predict that Arg-2 of α-tubulin forms hydrogen bonds with the GTPase domain of β-tubulin, and structural modeling suggests that these contacts are interrupted in tor2. Consistent with this, we found that microtubule dynamicity is reduced in the tor2 background. We investigated the developmental origin of the helical growth phenotype using tor2. One hypothesis predicts that cell division patterns cause helical organ growth in Arabidopsis thaliana mutants. However, cell division patterns of tor2 root tips appear normal. Experimental uncoupling of cell division and expansion suggests that helical organ growth is based on cell elongation defects only. Another hypothesis is that twisting is due to inequalities in expansion of epidermal and cortical tissues. However, freely growing leaf trichomes of tor2 mutants show right-handed twisting and cortical microtubules form left-handed helices as early as the unbranched stage of trichome development. Trichome twisting is inverted in double mutants with tor3, a left-handed mutant. Single tor2 suspension cells also exhibit handed twisting. Thus, twisting of tor2 mutant organs appears to be a higher-order expression of the helical expansion of individual cells.     

Long-term inhibition by auxin of leaf blade expansion in bean and Arabidopsis

The role of auxin in controlling leaf expansion remains unclear. Experimental increases to normal auxin levels in expanding leaves have shown conflicting results, with both increases and decreases in leaf growth having been measured. Therefore, the effects of both auxin application and adjustment of endogenous leaf auxin levels on midrib elongation and final leaf size (fresh weight and area) were examined in attached primary monofoliate leaves of the common bean (Phaseolus vulgaris) and in early Arabidopsis rosette leaves. Aqueous auxin application inhibited long-term leaf blade elongation. Bean leaves, initially 40 to 50 mm in length, treated once with alpha-naphthalene acetic acid (1.0 mm), were, after 6 d, approximately 80% the length and weight of controls. When applied at 1.0 and 0.1 mm, alpha-naphthalene acetic acid significantly inhibited long-term leaf growth. The weak auxin, beta-naphthalene acetic acid, was effective at 1.0 mm; and a weak acid control, benzoic acid, was ineffective. Indole-3-acetic acid (1 microm, 10 microm, 0.1 mm, and 1 mm) required daily application to be effective at any concentration. Application of the auxin transport inhibitor, 1-N-naphthylphthalamic acid (1% [w/w] in lanolin), to petioles also inhibited long-term leaf growth. This treatment also was found to lead to a sustained elevation of leaf free indole-3-acetic acid content relative to untreated control leaves. Auxin-induced inhibition of leaf growth appeared not to be mediated by auxin-induced ethylene synthesis because growth inhibition was not rescued by inhibition of ethylene synthesis. Also, petiole treatment of Arabidopsis with 1-N-naphthylphthalamic acid similarly inhibited leaf growth of both wild-type plants and ethylene-insensitive ein4 mutants.     

The role of auxin-binding protein 1 in the expansion of tobacco leaf cells

Tobacco leaf was used to investigate the mechanism of action of auxin-binding protein 1 (ABP1). The distributions of free auxin, ABP1, percentage of leaf nuclei in G2 and the amount of auxin-inducible growth were each determined in control tobacco leaves and leaves over-expressing Arabidopsis ABP1. These parameters were compared with growth of tobacco leaves, measured both spatially and temporally throughout the entire expansion phase. Within a defined window of leaf development, juvenile leaf cells that inducibly expressed Arabidopsis ABP1 prematurely advanced nuclei to the G2 phase. The ABP1-induced increase in cell expansion occured before the advance to the G2 phase, indicating that the ABP1-induced G2 phase advance is an indirect effect of cell expansion. The level of ABP1 was highest at the position of maximum cell expansion, maximum auxin-inducible growth and where the free auxin level was the lowest. In contrast, the position of maximum cell division correlated with higher auxin levels and lower ABP1 levels. Consistent with the correlations observed in leaves, tobacco cells (BY-2) in culture displayed two dose-dependent responses to auxin. At a low auxin concentration, cells expanded, while at a relatively higher concentration, cells divided and incorporated [3H]-thymidine. Antisense suppression of ABP1 in these cells dramatically reduced cell expansion with negligible effect on cell division. Taken together, the data suggest that ABP1 acts at a relatively low level of auxin to mediate cell expansion, whereas high auxin levels stimulate cell division via an unidentified receptor.     

Analysis of leaf development in fugu mutants of Arabidopsis reveals three compensation modes that modulate cell expansion in determinate organs

In multicellular organisms, the coordination of cell proliferation and expansion is fundamental for proper organogenesis, yet the molecular mechanisms involved in this coordination are largely unexplored. In plant leaves, the existence of this coordination is suggested by compensation, in which a decrease in cell number triggers an increase in mature cell size. To elucidate the mechanisms of compensation, we isolated five new Arabidopsis (Arabidopsis thaliana) mutants (fugu1-fugu5) that exhibit compensation. These mutants were characterized together with angustifolia3 (an3), erecta (er), and a KIP-RELATED PROTEIN2 (KRP2) overexpressor, which were previously reported to exhibit compensation. Time-course analyses of leaf development revealed that enhanced cell expansion in fugu2-1, fugu5-1, an3-4, and er-102 mutants is induced postmitotically, indicating that cell enlargement is not caused by the uncoupling of cell division from cell growth. In each of the mutants, either the rate or duration of cell expansion was selectively enhanced. In contrast, we found that enhanced cell expansion in KRP2 overexpressor occurs during cell proliferation. We further demonstrated that enhanced cell expansion occurs in cotyledons with dynamics similar to that in leaves. In contrast, cell expansion was not enhanced in roots even though they exhibit decreased cell numbers. Thus, compensation was confirmed to occur preferentially in determinate organs. Flow cytometric analyses revealed that increases in ploidy level are not always required to trigger compensation, suggesting that compensation is only partially mediated by ploidy-dependent processes. Our results suggest that compensation reflects an organ-wide coordination of cell proliferation and expansion in determinate organs, and involves at least three different expansion pathways.     

Establishment of polarity in lateral organs of plants

BACKGROUND: Asymmetric development of plant lateral organs initiates by partitioning of organ primordia into distinct domains along their adaxial/abaxial axis. A recent model proposes that a meristem-born signal, acting in a concentration-dependent manner, differentially activates PHABULOSA-like genes, which in turn suppress abaxial-promoting factors. As yet, no abaxial factors have been identified that when compromised give rise to adaxialized organs. RESULTS: Single mutants in either of the closely related genes KANADI1 (KAN1) or KANADI2 (KAN2) have little or no effect on plant morphology. However, in kan1 kan2 double mutant plants, there is a replacement of abaxial cell types by adaxial ones in most lateral organs. The alterations in polarity establishment are associated with expansion in the expression domain of the PHB-like genes and reduction in the expression of the previously described abaxial-promoting YABBY genes. Ectopic expression of either of the KANADI genes throughout leaf primordia results in dramatic transformation of adaxial cell types into abaxial ones, failure of lateral blade expansion, and vascular tissue formation. CONCLUSION: The phenotypes of KANADI loss- and gain-of-function alleles suggest that fine regulation of these genes is at the core of polarity establishment. As such, they are likely to be targets of the PHB-mediated meristem-born signaling that patterns lateral organ primordia. PHB-like genes and the abaxial-promoting KANADI and YABBY genes appear to be expressed throughout primordia anlagen before becoming confined to their corresponding domains as primordia arise. This suggests that the establishment of polarity in plant lateral organs occurs via mutual repression interactions between ab/ad factors after primordium emergence, consistent with the results of classical dissection experiments.     

YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development

During leaf development, the formation of leaf adaxial-abaxial polarity at the primordium stage is crucial for subsequent leaf expansion. However, little is known about the genetic control from polarity establishment to blade outgrowth. The leaf margin, comprising elongated margin cells and hydathodes, is thought to affect leaf expansion. Here, we show that mutants with defective leaf polarity or with loss of function in the multiple auxin-biosynthetic YUCCA (YUC) genes exhibited a similar abnormal leaf margin and less-expanded leaves. Leaf margins of these mutants contained fewer hydathodes and an increased number of cell patches in which the patterns of epidermal cells resembled those of hydathodes. The previously characterized leaf-abaxialized asymmetric leaves2 (as2) revoluta (rev) and leaf-adaxialized kanadi1 (kan1) kan2 double mutants both produce finger-shaped, hydathode-like protrusions on adaxial and abaxial leaf surfaces, respectively. YUCs are required for formation of the protrusions, as those produced by as2 rev and kan1 kan2 were absent in the yuc1 yuc2 yuc4 triple mutant background. Expressions of YUC1, YUC2, and YUC4 were spatially regulated in the leaf, being associated with hydathodes in wild-type leaves and protrusions on as2 rev and kan1 kan2 leaves. In addition, inhibition of auxin transport by treatment of seedlings with N-(1-naphtyl) phtalamic acid or disruption of the auxin gradient by transforming plants with the 35S:YUC1 construct also blocked leaf margin development. Collectively, our data show that expressions of YUCs in the leaf respond to the adaxial-abaxial juxtaposition, and that the activities of auxin mediate leaf margin development, which subsequently promotes blade outgrowth.