Genetic Interaction between Arabidopsis SUR2/CYP83B1 and GNOM Indicates the Importance of Stabilizing Local Auxin Accumulation in Lateral Root Initiation.

Lateral root (LR) formation is an important developmental event for the establishment of the root system in most vascular plants. In Arabidopsis thaliana, the fewer roots (fwr) mutation in the GNOM gene, encoding a guanine nucleotide exchange factor of ADP ribosylation factor that regulates vesicle trafficking, severely inhibits LR formation. Local accumulation of auxin response for LR initiation is severely affected in fwr. To better understand how local accumulation of auxin response for LR initiation is regulated, we identified a mutation, fewer roots suppressor1 (fsp1), that partially restores LR formation in fwr. The gene responsible for fsp1 was identified as SUPERROOT2 (SUR2), encoding CYP83B1 that positions at the metabolic branch point in the biosynthesis of auxin/indole-3-acetic acid (IAA) and indole glucosinolate. The fsp1 mutation increases both endogenous IAA levels and the number of the sites where auxin response locally accumulates prior to LR formation in fwr. SUR2 is expressed in the pericycle of the differentiation zone and in the apical meristem in roots. Time-lapse imaging of the auxin response revealed that local accumulation of auxin response is more stable in fsp1. These results suggest that SUR2/CYP83B1 affects LR founder cell formation at the xylem pole pericycle cells where auxin accumulates. Analysis of the genetic interaction between SUR2 and GNOM indicates the importance of stabilization of local auxin accumulation sites for LR initiation.

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.

Distinct gibberellin functions during and after grapevine bud dormancy release.

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It has been hypothesized that (i) abscisic acid (ABA) represses bud-meristem activity; (ii) perturbation of respiration induces an interplay between ethylene and ABA metabolism, which leads to removal of repression; and (iii) gibberellin (GA)-mediated growth is resumed. The first two hypothesis have been formally supported. The current study examines the third hypothesis regarding the potential involvement of GA in dormancy release. We found that during natural dormancy induction, levels of VvGA3ox, VvGA20ox, and VvGASA2 transcripts and of GA1 were decreased. However, during dormancy release, expression of these genes was enhanced, accompanied by decreased expression of the bud-expressed GA-deactivating VvGA2ox. Despite indications for its positive role during natural dormancy release, GA application had inhibitory effects on bud break. Hydrogen cyanamide up-regulated VvGA2ox and down-regulated VvGA3ox and VvGA20ox expression, reduced GA1 levels, and partially rescued the negative effect of GA. GA had an inhibitory effect only when applied simultaneously with bud-forcing initiation. Given these results, we hypothesize that during initial activation of the dormant bud meristem, the level of GA must be restricted, but after meristem activation an increase in its level serves to enhance primordia regrowth.

The reduction in maize leaf growth under mild drought affects the transition between cell division and cell expansion and cannot be restored by elevated gibberellic acid levels.

Growth is characterized by the interplay between cell division and cell expansion, two processes that occur separated along the growth zone at the maize leaf. To gain further insight into the transition between cell division and cell expansion, conditions were investigated in which the position of this transition zone was positively or negatively affected. High levels of gibberellic acid (GA) in plants overexpressing the GA biosynthesis gene GA20-OXIDASE (GA20OX-1OE ) shifted the transition zone more distally, whereas mild drought, which is associated with lowered GA biosynthesis, resulted in a more basal positioning. However, the increased levels of GA in the GA20OX-1OE line were insufficient to convey tolerance to the mild drought treatment, indicating that another mechanism in addition to lowered GA levels is restricting growth during drought. Transcriptome analysis with high spatial resolution indicated that mild drought specifically induces a reprogramming of transcriptional regulation in the division zone. 'Leaf Growth Viewer' was developed as an online searchable tool containing the high-resolution data.

Abnormal Endogenous Repression of GA Signaling in a Seedless Table Grape Cultivar with High Berry Growth Response to GA Application.

Gibberellin (GA) application is routinely used in the table grape industry to increase berry size and cluster length. Although grapevine cultivars show a wide range of growth responsiveness to GA3 application, the reasons for these differences is unclear. To shed light on this issue, two commercial grapevine cultivars with contrasting berry response to GA were selected for comparative analysis, in which we tested if the differences in response: (1) is organ-specific or cultivar-related; (2) will be reflected in qualitative/quantitative differences in transcripts/proteins of central components of GA metabolism and signaling and levels of GA metabolites. Our results showed that in addition to the high response of its berries to GA, internodes and rachis of cv. Black finger (BF) presented a greater growth response compared to that of cv. Spring blush (SB). In agreement, the results exposed significant quantitative differences in GA signaling components in several organs of both cultivars. Exceptionally higher level of all three functional VvDELLA proteins was recorded in young BF organs, accompanied by elevated VvGID1 expression and lower VvSLY1b transcripts. Absence of seed traces, low endogenous GA quantities and lower expression of VvGA20ox4 and VvGA3ox3 were also recorded in berries of BF. Our results raise the hypothesis that, in young organs of BF, low expression of VvSLY1b may be responsible for the massive accumulation of VvDELLA proteins, which then leads to elevated VvGID1 levels. This integrated analysis suggests causal relationship between endogenous mechanisms leading to anomalous GA signaling repression in BF, manifested by high quantities of VvDELLA proteins, and greater growth response to GA application.

Jasmonates are induced by the PAMP flg22 but not the cell death-inducing elicitor Harpin in Vitis rupestris.

Plants employ two layers of defence that differ with respect to cell death: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI). In our previous work, we have comparatively mapped the molecular events in a cell system derived from the wild American grape Vitis rupestris, where cell death-independent defence can be triggered by PAMP flg22, whereas the elicitor Harpin activates a cell death-related ETI-like response. Both defence responses overlapped with respect to early events, such as calcium influx, apoplastic alkalinisation, oxidative burst, mitogen-activated protein kinase (MAPK) signalling, activation of defence-related genes and accumulation of phytoalexins. However, timing and amplitude of early signals differed. In the current study, we address the role of jasmonates (JAs) as key signalling compounds in hypersensitive cell death. We find, in V. rupestris, that jasmonic acid and its bioactive conjugate jasmonoyl-isoleucine (JA-Ile) rapidly accumulate in response to flg22 but not in response to Harpin. However, Harpin can induce programmed cell death, whereas exogenous methyl jasmonate (MeJA) fails to do so, although both signals induce a similar response of defence genes. Also in a second cell line from V. vinifera cv. 'Pinot Noir', where Harpin cannot activate cell death and where flg22 fails to induce JA and JA-Ile, defence genes are activated in a similar manner. These findings indicate that the signal pathway culminating in cell death must act independently from the events culminating in the accumulation of toxic stilbenes.

Analysis of plant hormone profiles in response to moderate dehydration stress.

Plant responses to dehydration stress are mediated by highly complex molecular systems involving hormone signaling and metabolism, particularly the major stress hormone abscisic acid (ABA) and ABA-dependent gene expression. To understand the roles of plant hormones and their interactions during dehydration, we analyzed the plant hormone profiles with respect to dehydration responses in Arabidopsis thaliana wild-type (WT) plants and ABA biosynthesis mutants (nced3-2). We developed a procedure for moderate dehydration stress, and then investigated temporal changes in the profiles of ABA, jasmonic acid isoleucine (JA-Ile), salicylic acid (SA), cytokinin (trans-zeatin, tZ), auxin (indole-acetic acid, IAA), and gibberellin (GA4 ), along with temporal changes in the expression of key genes involved in hormone biosynthesis. ABA levels increased in a bi-phasic pattern (at the early and late phases) in response to moderate dehydration stress. JA-Ile levels increased slightly in WT plants and strongly increased in nced3-2 mutant plants at 72 h after the onset of dehydration. The expression profiles of dehydration-inducible genes displayed temporal responses in an ABA-dependent manner. The early phase of ABA accumulation correlated with the expression of touch-inducible genes and was independent of factors involved in the major ABA regulatory pathway, including the ABA-responsive element-binding (AREB/ABF) transcription factor. JA-Ile, SA, and tZ were negatively regulated during the late dehydration response phase. Transcriptome analysis revealed important roles for hormone-related genes in metabolism and signaling during dehydration-induced plant responses.

AtSWEET13 and AtSWEET14 regulate gibberellin-mediated physiological processes.

Transmembrane transport of plant hormones is required for plant growth and development. Despite reports of a number of proteins that can transport the plant hormone gibberellin (GA), the mechanistic basis for GA transport and the identities of the transporters involved remain incomplete. Here, we provide evidence that Arabidopsis SWEET proteins, AtSWEET13 and AtSWEET14, which are members of a family that had previously been linked to sugar transport, are able to mediate cellular GA uptake when expressed in yeast and oocytes. A double sweet13 sweet14 mutant has a defect in anther dehiscence and this phenotype can be reversed by exogenous GA treatment. In addition, sweet13 sweet14 exhibits altered long distant transport of exogenously applied GA and altered responses to GA during germination and seedling stages. These results suggest that AtSWEET13 and AtSWEET14 may be involved in modulating GA response in Arabidopsis.

The ERF11 Transcription Factor Promotes Internode Elongation by Activating Gibberellin Biosynthesis and Signaling.

The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in plants. Previous studies show that GA activates its signaling pathway by inducing rapid degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel positive regulator of both GA biosynthesis and GA signaling for internode elongation. Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6 AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of ERF/AP2 transcription factors in Arabidopsis (Arabidopsis thaliana). Overexpression of AtERF11 resulted in elevated bioactive GA levels by up-regulating expression of GA3ox1 and GA20ox genes. Hypocotyl elongation assays further showed that overexpression of AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 mutants displayed reduced GA response. In addition, yeast two-hybrid, coimmunoprecipitation, and transient expression assays showed that AtERF11 enhances GA signaling by antagonizing the function of DELLA proteins via direct protein-protein interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels of another phytohormone ethylene in the growing stem, consistent with recent finding showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a dual role in promoting internode elongation by inhibiting ethylene biosynthesis and activating GA biosynthesis and signaling pathways.

Highly Sprouting-Tolerant Wheat Grain Exhibits Extreme Dormancy and Cold Imbibition-Resistant Accumulation of Abscisic Acid.

Pre-harvest sprouting (PHS) of wheat (Triticum aestivum L.) grains induces hydrolyzing enzymes such as α-amylase, which considerably decreases wheat product quality. PHS occurs when cool and wet weather conditions before harvest break dormancy and induce grain germination. In this study, we used PHS-tolerant varieties, Gifu-komugi (Gifu) and OS38, to characterize the mechanisms of both dormancy breakage and dormancy maintenance at low temperatures. Physiologically mature Gifu grains exhibited dormancy after imbibition at 20°C, but germinated at 15°C. In contrast, OS38 grains remained dormant even at temperatures as low as 5°C. Embryo half-grains cut out from the dormant Gifu grains germinated by imbibition at 20°C, similar to conventional varieties worldwide. However, OS38 embryo half-grains were still dormant. Hormonome and pharmacological analyses suggested that ABA and gibberellin metabolism are important for temperature-dependent dormancy maintenance and breakage. Imbibition at 15°C decreased ABA levels but increased gibberellin levels in embryos of freshly harvested Gifu grains. Additionally, low temperatures induced expression of the ABA catabolism genes,TaABA8' OH1 and TaABA8' OH2, and the gibberellin biosynthesis gene,TaGA3ox2, in the embryos. However, in embryos of freshly harvested OS38 grains, ABA levels were increased while gibberellin levels were suppressed at 15°C. In these dormant embryos, low temperatures induced the TaNCED ABA biosynthesis genes, but suppressed TaABA8' OH2 and TaGA3ox2.These results show that the regulatory mechanism influencing the expression of ABA and gibberellin metabolism genes may be critical for dormancy maintenance and breakage at low temperatures. Our findings should help improve PHS-resistant wheat breeding programs.

Enhancement of hypocotyl elongation by LOV KELCH PROTEIN2 production is mediated by auxin and phytochrome-interacting factors in Arabidopsis thaliana.

KEY MESSAGE: Auxin and two phytochrome-interacting factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, play crucial roles in the enhancement of hypocotyl elongation in transgenic Arabidopsis thaliana plants that overproduce LOV KELCH PROTEIN2 (LKP2). LOV KELCH PROTEIN2 (LKP2) is a positive regulator of hypocotyl elongation under white light in Arabidopsis thaliana. In this study, using microarray analysis, we compared the gene expression profiles of hypocotyls of wild-type Arabidopsis (Columbia accession), a transgenic line that produces green fluorescent protein (GFP), and two lines that produce GFP-tagged LKP2 (GFP-LKP2). We found that, in GFP-LKP2 hypocotyls, 775 genes were up-regulated, including 36 auxin-responsive genes, such as 27 SMALL AUXIN UP RNA (SAUR) and 6 AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) genes, and 21 genes involved in responses to red or far-red light, including PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5; and 725 genes were down-regulated, including 15 flavonoid biosynthesis genes. Hypocotyls of GFP-LKP2 seedlings, but not cotyledons or roots, contained a higher level of indole-3-acetic acid (IAA) than those of control seedlings. Auxin inhibitors reduced the enhancement of hypocotyl elongation in GFP-LKP2 seedlings by inhibiting the increase in cortical cell number and elongation of the epidermal and cortical cells. The enhancement of hypocotyl elongation was completely suppressed in progeny of the crosses between GFP-LKP2 lines and dominant gain-of-function auxin-resistant mutants (axr2-1 and axr3-1) or loss-of-function mutants pif4, pif5, and pif4 pif5. Our results suggest that the enhancement of hypocotyl elongation in GFP-LKP2 seedlings is due to the elevated level of IAA and to the up-regulated expression of PIF4 and PIF5 in hypocotyls.

A balanced JA/ABA status may correlate with adaptation to osmotic stress in Vitis cells.

Water-related stress is considered a major type of plant stress. Osmotic stress, in particular, represents the common part of all water-related stresses. Therefore, plants have evolved different adaptive mechanisms to cope with osmotic-related disturbances. In the current work, two grapevine cell lines that differ in their osmotic adaptability, Vitis rupestris and Vitis riparia, were investigated under mannitol-induced osmotic stress. To dissect signals that lead to adaptability from those related to sensitivity, osmotic-triggered responses with respect to jasmonic acid (JA) and its active form JA-Ile, abscisic acid (ABA), and stilbene compounds, as well as the expression of their related genes were observed. In addition, the transcript levels of the cellular homeostasis gene NHX1 were examined. The data are discussed with a hypothesis suggesting that a balance of JA and ABA status might correlate with cellular responses, either guiding cells to sensitivity or to progress toward adaptation.

Binding of GID1 to DELLAs promotes dissociation of GAF1 from DELLA in GA dependent manner.

Gibberellins (GAs) are important phytohormones for plant growth and development. DELLAs are members of the plant-specific GRAS protein family and act as repressors of GA signaling. DELLAs are rapidly degraded in the presence of GAs. GA-GID1-DELLA complexes are recognized and ubiquitinated by the SCF(SLY) complex. The sleepy1 (sly1) F-box mutant exhibits dwarfism and low-germination phenotypes due to high accumulation of DELLAs. Overexpression of GID1 in the sly1 mutant partially rescues these phenotypes without degradation of DELLAs suggesting that proteolysis independent regulation of DELLAs exists in GA signaling. But the molecular mechanisms of non-proteolytic regulation of DELLA are largely unknown. Recently we identified a DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 also interacts with co-repressor TOPLESS RELATED (TPR) in nuclei. DELLAs and TPR act as coactivator and corepressor of GAF1, respectively. GAs converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. The overexpression of ΔPAM, lacking of DELLAs binding region of GAF1, partially rescue dwarf phenotypes of GA deficient or GA insensitive mutant. In this study, we investigate the relationship between non-proteolytic regulation of DELLAs and GA signaling via DELLA-GAF1 complex using modified yeast two-hybrid system.

The control of tomato fruit elongation orchestrated by sun, ovate and fs8.1 in a wild relative of tomato.

Within the cultivated tomato germplasm, sun, ovate and fs8.1 are the three predominant QTLs controlling fruit elongation. Although SUN and OVATE have been cloned, their role in plant growth and development are not well understood. To compare and contrast the effects of the three QTLs in a homogeneous background, we developed near isogenic lines (NILs) in the wild species Solanum pimpinellifolium LA1589 background. We carried out detailed morphological characterization of reproductive and vegetative organs in the single, double and triple NILs and determined the epistatic interactions of the three loci affecting fruit shape. The phenotypic evaluations demonstrated that the three loci regulate unique aspects of ovary and fruit elongation and in different temporal manners. The strongest effect on organ shape was caused by sun. In addition to fruit shape, sun also affected leaf and sepal elongation and stem thickness. The synergistic interaction between sun and ovate or fs8.1 suggested that the pathways involving SUN, OVATE and the gene(s) underlying fs8.1 may converge at a common node. The results of an extensive profiling analysis suggested that the degree of fruit elongation was not related to the accumulation of any of the classical hormones.

Distinct Characteristics of Indole-3-Acetic Acid and Phenylacetic Acid, Two Common Auxins in Plants.

The phytohormone auxin plays a central role in many aspects of plant growth and development. IAA is the most studied natural auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin for >40 years, but its role in plant growth and development remains unclear. In this study, we show that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. PAA is widely distributed in vascular and non-vascular plants. Although the biological activities of PAA are lower than those of IAA, the endogenous levels of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin-responsive genes through the TIR1/AFB pathway in Arabidopsis. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA (YUC) genes increases PAA metabolite levels in Arabidopsis, indicating that YUC flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by different types of auxins.

Network Analyses Reveal Shifts in Transcript Profiles and Metabolites That Accompany the Expression of SUN and an Elongated Tomato Fruit.

SUN controls elongated tomato (Solanum lycopersicum) shape early in fruit development through changes in cell number along the different axes of growth. The gene encodes a member of the IQ domain family characterized by a calmodulin binding motif. To gain insights into the role of SUN in regulating organ shape, we characterized genome-wide transcriptional changes and metabolite and hormone accumulation after pollination and fertilization in wild-type and SUN fruit tissues. Pericarp, seed/placenta, and columella tissues were collected at 4, 7, and 10 d post anthesis. Pairwise comparisons between SUN and the wild type identified 3,154 significant differentially expressed genes that cluster in distinct gene regulatory networks. Gene regulatory networks that were enriched for cell division, calcium/transport, lipid/hormone, cell wall, secondary metabolism, and patterning processes contributed to profound shifts in gene expression in the different fruit tissues as a consequence of high expression of SUN. Promoter motif searches identified putative cis-elements recognized by known transcription factors and motifs related to mitotic-specific activator sequences. Hormone levels did not change dramatically, but some metabolite levels were significantly altered, namely participants in glycolysis and the tricarboxylic acid cycle. Also, hormone and primary metabolite networks shifted in SUN compared with wild-type fruit. Our findings imply that SUN indirectly leads to changes in gene expression, most strongly those involved in cell division, cell wall, and patterning-related processes. When evaluating global coregulation in SUN fruit, the main node represented genes involved in calcium-regulated processes, suggesting that SUN and its calmodulin binding domain impact fruit shape through calcium signaling.

Mystery in genetics: PUB4 gives a clue to the complex mechanism of CLV signaling pathway in the shoot apical meristem.

Postembryonic growth and development in higher plants are ultimately reliant on the activity of meristems, where the cells divide frequently to provide source cells for new organs and tissues while in part maintain their pluripotent nature as stem cells. The shoot apical meristem (SAM) is maintained throughout the life of plants and responsible for the development of all areal tissues. In Arabidopsis thaliana, the size of SAM is controlled by a peptide ligand, CLAVATA3 (CLV3). Previously, genetic studies have identified several genes that function downstream of CLV3, many of which, intriguingly, encode receptors. Recently we identified an E3 ubiquitin ligase, PLANT U-BOX 4 (PUB4), as a key regulatory component of root meristem maintenance that functions downstream of an exogenous synthetic CLV3 peptide. Here, we report an additional function of PUB4 in the SAM.

Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant hormones.

NRT1/PTR FAMILY (NPF) proteins were originally identified as nitrate or di/tri-peptide transporters. Recent studies revealed that this transporter family also transports the plant hormones auxin (indole-3-acetic acid), abscisic acid (ABA), and gibberellin (GA), as well as secondary metabolites (glucosinolates). We developed modified yeast two-hybrid systems with receptor complexes for GA and jasmonoyl-isoleucine (JA-Ile), to detect GA and JA-Ile transport activities of proteins expressed in the yeast cells. Using these GA and JA-Ile systems as well as the ABA system that we had introduced previously, we determined the capacities of Arabidopsis NPFs to transport these hormones. Several NPFs induced the formation of receptor complexes under relatively low hormone concentrations. Hormone transport activities were confirmed for some NPFs by direct analysis of hormone uptake of yeast cells by liquid chromatography-tandem mass spectrometry. Our results suggest that at least some NPFs could function as hormone transporters.