PIF4 enhances the expression of SAUR genes to promote growth in response to nitrate.

Nitrate supply is fundamental to support shoot growth and crop performance, but the associated increase in stem height exacerbates the risks of lodging and yield losses. Despite their significance for agriculture, the mechanisms involved in the promotion of stem growth by nitrate remain poorly understood. Here, we show that the elongation of the hypocotyl of Arabidopsis thaliana, used as a model, responds rapidly and persistently to upshifts in nitrate concentration, rather than to the nitrate level itself. The response occurred even in shoots dissected from their roots and required NITRATE TRANSPORTER 1.1 (NRT1.1) in the phosphorylated state (but not NRT1.1 nitrate transport capacity) and NIN-LIKE PROTEIN 7 (NLP7). Nitrate increased PHYTOCHROME INTERACTING FACTOR 4 (PIF4) nuclear abundance by posttranscriptional mechanisms that depended on NRT1.1 and phytochrome B. In response to nitrate, PIF4 enhanced the expression of numerous SMALL AUXIN-UP RNA (SAUR) genes in the hypocotyl. The growth response to nitrate required PIF4, positive and negative regulators of its activity, including AUXIN RESPONSE FACTORs, and SAURs. PIF4 integrates cues from the soil (nitrate) and aerial (shade) environments adjusting plant stature to facilitate access to light.

High levels of auxin signalling define the stem-cell organizer of the vascular cambium.

Wood, a type of xylem tissue, originates from cell proliferation of the vascular cambium. Xylem is produced inside, and phloem outside, of the cambium1. Morphogenesis in plants is typically coordinated by organizer cells that direct the adjacent stem cells to undergo programmed cell division and differentiation. The location of the vascular cambium stem cells and whether the organizer concept applies to the cambium are currently unknown2. Here, using lineage-tracing and molecular genetic studies in the roots of Arabidopsis thaliana, we show that cells with a xylem identity direct adjacent vascular cambial cells to divide and function as stem cells. Thus, these xylem-identity cells constitute an organizer. A local maximum of the phytohormone auxin, and consequent expression of CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) transcription factors, promotes xylem identity and cellular quiescence of the organizer cells. Additionally, the organizer maintains phloem identity in a non-cell-autonomous fashion. Consistent with this dual function of the organizer cells, xylem and phloem originate from a single, bifacial stem cell in each radial cell file, which confirms the classical theory of a uniseriate vascular cambium3. Clones that display high levels of ectopically activated auxin signalling differentiate as xylem vessels; these clones induce cell divisions and the expression of cambial and phloem markers in the adjacent cells, which suggests that a local auxin-signalling maximum is sufficient to specify a stem-cell organizer. Although vascular cambium has a unique function among plant meristems, the stem-cell organizer of this tissue shares features with the organizers of root and shoot meristems.

SAUR63 stimulates cell growth at the plasma membrane.

In plants, regulated cell expansion determines organ size and shape. Several members of the family of redundantly acting Small Auxin Up RNA (SAUR) proteins can stimulate plasma membrane (PM) H+-ATPase proton pumping activity by inhibiting PM-associated PP2C.D phosphatases, thereby increasing the PM electrochemical potential, acidifying the apoplast, and stimulating cell expansion. Similarly, Arabidopsis thaliana SAUR63 was able to increase growth of various organs, antagonize PP2C.D5 phosphatase, and increase H+-ATPase activity. Using a gain-of-function approach to bypass genetic redundancy, we dissected structural requirements for SAUR63 growth-promoting activity. The divergent N-terminal domain of SAUR63 has a predicted basic amphipathic α-helix and was able to drive partial PM association. Deletion of the N-terminal domain decreased PM association of a SAUR63 fusion protein, as well as decreasing protein level and eliminating growth-promoting activity. Conversely, forced PM association restored ability to promote H+-ATPase activity and cell expansion, indicating that SAUR63 is active when PM-associated. Lipid binding assays and perturbations of PM lipid composition indicate that the N-terminal domain can interact with PM anionic lipids. Mutations in the conserved SAUR domain also reduced PM association in root cells. Thus, both the N-terminal domain and the SAUR domain may cooperatively mediate the SAUR63 PM association required to promote growth.

Modulating auxin response stabilizes tomato fruit set.

Fruit formation depends on successful fertilization and is highly sensitive to weather fluctuations that affect pollination. Auxin promotes fruit initiation and growth following fertilization. Class A auxin response factors (Class A ARFs) repress transcription in the absence of auxin and activate transcription in its presence. Here, we explore how multiple members of the ARF family regulate fruit set and fruit growth in tomato (Solanum lycopersicum) and Arabidopsis thaliana, and test whether reduction of SlARF activity improves yield stability in fluctuating temperatures. We found that several tomato Slarf mutant combinations produced seedless parthenocarpic fruits, most notably mutants deficient in SlARF8A and SlARF8B genes. Arabidopsis Atarf8 mutants deficient in the orthologous gene had less complete parthenocarpy than did tomato Slarf8a Slarf8b mutants. Conversely, Atarf6 Atarf8 double mutants had reduced fruit growth after fertilization. AtARF6 and AtARF8 likely switch from repression to activation of fruit growth in response to a fertilization-induced auxin increase in gynoecia. Tomato plants with reduced SlARF8A and SlARF8B gene dosage had substantially higher yield than the wild type under controlled or ambient hot and cold growth conditions. In field trials, partial reduction in the SlARF8 dose increased yield under extreme temperature with minimal pleiotropic effects. The stable yield of the mutant plants resulted from a combination of early onset of fruit set, more fruit-bearing branches and more flowers setting fruits. Thus, ARF8 proteins mediate the control of fruit set, and relieving this control with Slarf8 mutations may be utilized in breeding to increase yield stability in tomato and other crops.

miR167 limits anther growth to potentiate anther dehiscence.

In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode auxin response transcription factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew larger than wild-type anthers did starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation.

SAUR proteins and PP2C.D phosphatases regulate H+-ATPases and K+ channels to control stomatal movements.

Activation of plasma membrane (PM) H+-ATPase activity is crucial in guard cells to promote light-stimulated stomatal opening, and in growing organs to promote cell expansion. In growing organs, SMALL AUXIN UP RNA (SAUR) proteins inhibit the PP2C.D2, PP2C.D5, and PP2C.D6 (PP2C.D2/5/6) phosphatases, thereby preventing dephosphorylation of the penultimate phosphothreonine of PM H+-ATPases and trapping them in the activated state to promote cell expansion. To elucidate whether SAUR-PP2C.D regulatory modules also affect reversible cell expansion, we examined stomatal apertures and conductances of Arabidopsis thaliana plants with altered SAUR or PP2C.D activity. Here, we report that the pp2c.d2/5/6 triple knockout mutant plants and plant lines overexpressing SAUR fusion proteins exhibit enhanced stomatal apertures and conductances. Reciprocally, saur56 saur60 double mutants, lacking two SAUR genes normally expressed in guard cells, displayed reduced apertures and conductances, as did plants overexpressing PP2C.D5. Although altered PM H+-ATPase activity contributes to these stomatal phenotypes, voltage clamp analysis showed significant changes also in K+ channel gating in lines with altered SAUR and PP2C.D function. Together, our findings demonstrate that SAUR and PP2C.D proteins act antagonistically to facilitate stomatal movements through a concerted targeting of both ATP-dependent H+ pumping and channel-mediated K+ transport.

Three Auxin Response Factors Promote Hypocotyl Elongation.

The hormone auxin regulates growth largely by affecting gene expression. By studying Arabidopsis (Arabidopsis thaliana) mutants deficient in AUXIN RESPONSE FACTORS (ARFs), we have identified three ARF proteins that are required for auxin-responsive hypocotyl elongation. Plants deficient in these factors have reduced responses to environmental conditions that increase auxin levels, including far-red-enriched light and high temperature. Despite having decreased auxin responses, the ARF-deficient plants responded to brassinosteroid and gibberellin, indicating that different hormones can act partially independently. Aux/IAA proteins, encoded by IAA genes, interact with ARF proteins to repress auxin response. Silencing expression of multiple IAA genes increased hypocotyl elongation, suggesting that Aux/IAA proteins modulate ARF activity in hypocotyls in a potential negative feedback loop.

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8.

Plant viral suppressors of RNA silencing induce developmental defects similar to those caused by mutations in genes involved in the microRNA pathway. A recent report has attributed viral suppressor-mediated developmental defects to up-regulation of AUXIN RESPONSE FACTOR 8 (ARF8), a target of miR167. The key piece of evidence was that the developmental defects in transgenic Arabidopsis (Arabidopsis thaliana) expressing viral suppressors were greatly alleviated in the F1 progeny of a cross with plants carrying the arf8-6 mutation. Arf8-6 is a SALK line T-DNA insertion mutant, a class of mutations prone to inducing transcriptional silencing of transgenes expressed from the 35S promoter. We have reinvestigated the role of ARF8 in viral suppressor-mediated developmental defects, using two independent arf8 mutations and the P1/HC-Pro potyviral suppressor of silencing. Progeny of a cross between P1/HC-Pro transgenic Arabidopsis and the arf8-6 T-DNA insertion mutant showed little effect on the P1/HC-Pro phenotype in the F1 generation, but almost all arf8-6/P1/HC-Pro progeny had lost the phenotype in the F2 generation. However, the loss of phenotype in the F2 generation was not correlated with the number of functional copies of the ARF8 gene. Instead, it reflected transcriptional silencing of the P1/HC-Pro transgene, as evidenced by a pronounced decrease in P1/HC-Pro mRNA and the appearance of 35S promoter small interfering RNAs. Furthermore, an independent loss-of-function point mutation, Arf8-8, had no detectable effects on P1/HC-Pro phenotype in either the F1 or F2 generations. Together, these data argue against the previously reported role of increased ARF8 expression in developmental defects caused by P1/HC-Pro.

ABCG transporters are required for suberin and pollen wall extracellular barriers in Arabidopsis.

Effective regulation of water balance in plants requires localized extracellular barriers that control water and solute movement. We describe a clade of five Arabidopsis thaliana ABCG half-transporters that are required for synthesis of an effective suberin barrier in roots and seed coats (ABCG2, ABCG6, and ABCG20) and for synthesis of an intact pollen wall (ABCG1 and ABCG16). Seed coats of abcg2 abcg6 abcg20 triple mutant plants had increased permeability to tetrazolium red and decreased suberin content. The root system of triple mutant plants was more permeable to water and salts in a zone complementary to that affected by the Casparian strip. Suberin of mutant roots and seed coats had distorted lamellar structure and reduced proportions of aliphatic components. Root wax from the mutant was deficient in alkylhydroxycinnamate esters. These mutant plants also had few lateral roots and precocious secondary growth in primary roots. abcg1 abcg16 double mutants defective in the other two members of the clade had pollen with defects in the nexine layer of the tapetum-derived exine pollen wall and in the pollen-derived intine layer. Mutant pollen collapsed at the time of anther desiccation. These mutants reveal transport requirements for barrier synthesis as well as physiological and developmental consequences of barrier deficiency.

In the absence of BYPASS1-related gene function, the bps signal disrupts embryogenesis by an auxin-independent mechanism.

Development is often coordinated by biologically active mobile compounds that move between cells or organs. Arabidopsis mutants with defects in the BYPASS1 (BPS1) gene overproduce an active mobile compound that moves from the root to the shoot and inhibits growth. Here, we describe two related Arabidopsis genes, BPS2 and BPS3. Analyses of single, double and triple mutants revealed that all three genes regulate production of the same mobile compound, the bps signal, with BPS1 having the largest role. The triple mutant had a severe embryo defect, including the failure to properly establish provascular tissue, the shoot meristem and the root meristem. Aberrant expression of PINFORMED1, DR5, PLETHORA1, PLETHORA2 and WUSCHEL-LIKE HOMEOBOX5 were found in heart-stage bps triple-mutant embryos. However, auxin-induced gene expression, and localization of the PIN1 auxin efflux transporter, were intact in bps1 mutants, suggesting that the primary target of the bps signal is independent of auxin response. Thus, the bps signal identifies a novel signaling pathway that regulates patterning and growth in parallel with auxin signaling, in multiple tissues and at multiple developmental stages.

A regulatory network for coordinated flower maturation.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs.

Down-regulation of AUXIN RESPONSE FACTORS 6 and 8 by microRNA 167 leads to floral development defects and female sterility in tomato.

Auxin regulates the expression of diverse genes that affect plant growth and development. This regulation requires AUXIN RESPONSE FACTORS (ARFs) that bind to the promoter regions of these genes. ARF6 and ARF8 in Arabidopsis thaliana are required to promote inflorescence stem elongation and late stages of petal, stamen, and gynoecium development. All seed plants studied thus far have ARF6 and ARF8 orthologues as well as the microRNA miR167, which targets ARF6 and ARF8. Whether these genes have broadly conserved roles in flower development is not known. To address this question, the effects of down-regulation of ARF6 and ARF8 were investigated through transgenic expression of Arabidopsis MIR167a in tomato, which diverged from Arabidopsis before the radiation of dicotyledonous plants approximately 90-112 million years ago. The transgenic tomato plants overexpressing MIR167a exhibited reductions in leaf size and internode length as well as shortened petals, stamens, and styles. More significantly, the transgenic plants were female-sterile as a result of failure of wild-type pollen to germinate on the stigma surface and/or to grow through the style. RNA-Seq analysis identified many genes with significantly altered expression patterns, including those encoding products with functions in 'transcription regulation', 'cell wall' and 'lipid metabolism' categories. Putative orthologues of a subset of these genes were also differentially expressed in Arabidopsis arf6 arf8 mutant flowers. These results thus suggest that ARF6 and ARF8 have conserved roles in controlling growth and development of vegetative and flower organs in dicots.

Arabidopsis SMALL AUXIN UP RNA63 promotes hypocotyl and stamen filament elongation.

Auxin regulates plant growth and development in part by activating gene expression. Arabidopsis thaliana SMALL AUXIN UP RNAs (SAURs) are a family of early auxin-responsive genes with unknown functionality. Here, we show that transgenic plant lines expressing artificial microRNA constructs (aMIR-SAUR-A or -B) that target a SAUR subfamily (SAUR61-SAUR68 and SAUR75) had slightly reduced hypocotyl and stamen filament elongation. In contrast, transgenic plants expressing SAUR63:GFP or SAUR63:GUS fusions had long hypocotyls, petals and stamen filaments, suggesting that these protein fusions caused a gain of function. SAUR63:GFP and SAUR63:GUS seedlings also accumulated a higher level of basipetally transported auxin in the hypocotyl than did wild-type seedlings, and had wavy hypocotyls and twisted inflorescence stems. Mutations in auxin efflux carriers could partially suppress some SAUR63:GUS phenotypes. In contrast, SAUR63:HA plants had wild-type elongation and auxin transport. SAUR63:GFP protein had a longer half-life than SAUR63:HA. Fluorescence imaging and microsomal fractionation studies revealed that SAUR63:GFP was localized mainly in the plasma membrane, whereas SAUR63:HA was present in both soluble and membrane fractions. Low light conditions increased SAUR63:HA protein turnover rate. These results indicate that membrane-associated Arabidopsis SAUR63 promotes auxin-stimulated organ elongation.

Genetic dissection of the auxin response network.

The expansion of gene families during evolution, which can generate functional overlap or specialization among their members, is a characteristic feature of signalling pathways in complex organisms. For example, families of transcriptional activators and repressors mediate responses to the plant hormone auxin. Although these regulators were identified more than 20 years ago, their overlapping functions and compensating negative feedbacks have hampered their functional analyses. Studies using loss-of-function approaches in basal land plants and gain-of-function approaches in angiosperms have in part overcome these issues but have still left an incomplete understanding. Here, we propose that renewed emphasis on genetic analysis of multiple mutants and species will shed light on the role of gene families in auxin response. Combining loss-of-function mutations in auxin-response activators and repressors can unravel complex outputs enabled by expanded gene families, such as fine-tuned developmental outcomes and robustness. Similar approaches and concepts may help to analyse other regulatory pathways whose components are also encoded by large gene families.

A gain-of-function mutation in IAA18 alters Arabidopsis embryonic apical patterning.

Lateral organ emergence in plant embryos and meristems depends on spatially coordinated auxin transport and auxin response. Here, we report the gain-of-function iaa18-1 mutation in Arabidopsis, which stabilizes the Aux/IAA protein IAA18 and causes aberrant cotyledon placement in embryos. IAA18 was expressed in the apical domain of globular stage embryos, and in the shoot apical meristem and adaxial domain of cotyledons of heart stage embryos. Mutant globular embryos had asymmetric PIN1:GFP expression in the apical domain, indicating that IAA18-1 disrupts auxin transport. Genetic interactions among iaa18-1, loss-of-function mutations in ARF (Auxin response factor) genes and ARF-overexpressing constructs suggest that IAA18-1 inhibits activity of MP/ARF5 and other ARF proteins in the apical domain. The iaa18-1 mutation also increased the frequency of rootless seedlings in mutant backgrounds in which auxin regulation of basal pole development was affected. These results indicate that apical patterning requires Aux/IAA protein turnover, and that apical domain auxin response also influences root formation.

Arabidopsis microRNA167 controls patterns of ARF6 and ARF8 expression, and regulates both female and male reproduction.

In flowering plants, diploid sporophytic tissues in ovules and anthers support meiosis and subsequent haploid gametophyte development. These analogous reproductive functions suggest that common mechanisms may regulate ovule and anther development. Two Arabidopsis Auxin Response Factors, ARF6 and ARF8, regulate gynoecium and stamen development in immature flowers. Wild-type pollen grew poorly in arf6 arf8 gynoecia, correlating with ARF6 and ARF8 expression in style and transmitting tract. ARF6 and ARF8 transcripts are cleavage targets of the microRNA miR167, and overexpressing miR167 mimicked arf6 arf8 phenotypes. Mutations in the miR167 target sites of ARF6 or ARF8 caused ectopic expression of these genes in domains of both ovules and anthers where miR167 was normally present. As a result, ovule integuments had arrested growth, and anthers grew abnormally and failed to release pollen. Thus, miR167 is essential for correct patterning of gene expression, and for fertility of both ovules and anthers. The essential patterning function of miR167 contrasts with cases from animals in which miRNAs reinforce or maintain transcriptionally established gene expression patterns.

AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2 regulate senescence and floral organ abscission in Arabidopsis thaliana.

In plants, both endogenous mechanisms and environmental signals regulate developmental transitions such as seed germination, induction of flowering, leaf senescence and shedding of senescent organs. Auxin response factors (ARFs) are transcription factors that mediate responses to the plant hormone auxin. We have examined Arabidopsis lines carrying T-DNA insertions in AUXIN RESPONSE FACTOR1 (ARF1) and ARF2 genes. We found that ARF2 promotes transitions between multiple stages of Arabidopsis development. arf2 mutant plants exhibited delays in several processes related to plant aging, including initiation of flowering, rosette leaf senescence, floral organ abscission and silique ripening. ARF2 expression was induced in senescing leaves. ARF2 regulated leaf senescence and floral organ abscission independently of the ethylene and cytokinin response pathways. arf1 mutations enhanced many arf2 phenotypes, indicating that ARF1 acts in a partially redundant manner with ARF2. However, unlike arf2 mutations, an arf1 mutation increased transcription of Aux/IAA genes in Arabidopsis flowers, supporting previous biochemical studies that indicated that ARF1 is a transcriptional repressor. Two other ARF genes, NPH4/ARF7 and ARF19, were also induced by senescence, and mutations in these genes enhanced arf2 phenotypes. NPH4/ARF7 and ARF19 function as transcriptional activators, suggesting that auxin may control senescence in part by activating gene expression.

NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation.

Auxin response factors (ARFs) bind auxin response promoter elements and mediate transcriptional responses to auxin. Five of the 22 ARF genes in Arabidopsis thaliana encode ARFs with glutamine-rich middle domains. Four of these can activate transcription and have been ascribed developmental functions. We show that ARF19, the fifth Q-rich ARF, also activates transcription. Mutations in ARF19 have little effect on their own, but in combination with mutations in NPH4/ARF7, encoding the most closely related ARF, they cause several phenotypes including a drastic decrease in lateral and adventitious root formation and a decrease in leaf cell expansion. These results indicate that auxin induces lateral roots and leaf expansion by activating NPH4/ARF7 and ARF19. Auxin induces the ARF19 gene, and NPH4/ARF7 and ARF19 together are required for expression of one of the arf19 mutant alleles, suggesting that a positive feedback loop regulates leaf expansion and/or lateral root induction.

Developmental specificity of auxin response by pairs of ARF and Aux/IAA transcriptional regulators.

The plant hormone auxin elicits many specific context-dependent developmental responses. Auxin promotes degradation of Aux/IAA proteins that prevent transcription factors of the auxin response factor (ARF) family from regulating auxin-responsive target genes. Aux/IAAs and ARFs are represented by large gene families in Arabidopsis. Here we show that stabilization of BDL/IAA12 or its sister protein IAA13 prevents MP/ARF5-dependent embryonic root formation whereas stabilized SHY2/IAA3 interferes with seedling growth. Although both bdl and shy2-2 proteins inhibited MP/ARF5-dependent reporter gene activation, shy2-2 was much less efficient than bdl to interfere with embryonic root initiation when expressed from the BDL promoter. Similarly, MP was much more efficient than ARF16 in this process. When expressed from the SHY2 promoter, both shy2-2 and bdl inhibited cell elongation and auxin-induced gene expression in the seedling hypocotyl. By contrast, gravitropism and auxin-induced gene expression in the root, which were promoted by functionally redundant NPH4/ARF7 and ARF19 proteins, were inhibited by shy2-2, but not by bdl protein. Our results suggest that auxin signals are converted into specific responses by matching pairs of coexpressed ARF and Aux/IAA proteins.