Auxin activity_ Past, present, and future1

180A merican Journal of Botany 102 (2): 180 –196 ,2015 ;h ttp:/// © 2015B otanical Society of AmericaA M E R IC A N J OU R N AL OFB OTA NY RESEARCH ARTICLEA JB CE NTE N N IAL RE VIE W T his A merican Journal of Botany centennial review consid-ers the history of auxin, with a focus on an A JB classic paperabout auxin activity by Kenneth Thimann and Charles Schneider titled “The relative activities of different auxins” ( T himann and Schneider, 1939 ). Since the publication of this seminal paper in 1939, many of its reported observations can be ex-plained using the knowledge gained about auxin biology in the last 75 yr. In this review, we describe the history of the auxin fi eld, discuss a signifi cant study published in the A meri-can Journal of Botany , summarize what we have learned in more recent years about auxin biology, and conclude with a discussion of unanswered questions in the fi eld. We are hope-ful that the next 100 yr of auxin research will reveal answers to these questions.T he term “auxin” is derived from the Greek word “auxein” meaning “to grow”. Discovery of auxinic compounds (both naturally occurring and synthetic auxins) was facilitated by the A vena test ( F ig. 1A ) developed by W ent (1928) . The A vena test consists of placing an agar block containing test compounds onone side of excised A vena sativa coleoptile tips and measuringthe ensuing curvature (reviewed in W ent and Thimann, 1937 ). Thus, the classic defi ning feature of auxinic compounds is elici-tation of a physiological response in the A vena test. The fi rst compounds to be considered auxins were auxin a (auxenotriolic acid) and auxin b (auxenolonic acid) ( K ögl et al., 1933 , 1934a , b ). However, as time went on, many laboratories were unable to isolate these compounds and they fell out of favor as potential auxins (reviewed in W ent and Thimann, 1937 ). For a modern critique of the history of auxin a and auxin b , we recommend W ildman (1997) . Indole-3-acetic acid (IAA or heteroauxin) was later favored as the strongest auxin because of its potency as a plant growth regulator; many additional compounds ( F ig. 2 ) were also being used as auxins, including indole-3-butyric acid (IBA), indole-3-propionic acid (IPrA), and naphthalene acetic acid (NAA), among others.A lthough IAA was recognized for its effective ability to in-duce cell expansion in plants, it was fi rst isolated, not from plants, but from human urine ( K ögl et al., 1934b ), yeast ( K ögl and Kostermans, 1934 ), and R hizopus suinus ( T himann, 1935 ). Although IAA was the standard to which other auxin compounds were compared in T himann and Schneider (1939) , at the time of their study, IAA was not believed to be a native plant hormone. The elusive auxin a was thought to be the auxin present in V icia faba ,Z ea mays , and A vena sativa (reviewed in W ent and Thimann, 1937 ), whereas IAA was believed to be the natural auxin in fungal species such as A spergillus ,R hizopus , and yeast (reviewed in W ent and Thimann, 1937 ). Indeed, IAA was not isolated from higher plants until the early 1940s ( H aagen-Smit et al., 1941 ). During this time, additional compounds with auxin1 Manuscript received 23 June 2014; revision accepted 9 January 2015.We thank S. Wyatt for the invitation to write this review and E. Frick, A. Muehler-Sherp, M. Michniewicz, D. Korasick, and two anonymous reviewers for critical comments on the manuscript. This work was supported by the National Science Foundation (DGE-1143954 to T.A.E.) and the National Institutes of Health (R00 G M089987 to L.C.S.). We apologize to anyone whose work we have overlooked (see F ig. 8 ). 2 A uthor for correspondence (e-mail: strader@) d oi:10.3732/ajb.1400285A UXIN ACTIVITY : P AST , PRESENT , AND FUTURE 1T ARA A . E NDERS A ND L UCIA C . S TRADER 2D epartment of Biology, Washington University in St. Louis, St. Louis, Missouri 63130 USA Long before its chemical identity was known, the phytohormone auxin was postulated to regulate plant growth. In the late 1800s, Sachs hypothesized that plant growth regulators, present in small amounts, move differentially throughout the plant to regulate growth. Concurrently, Charles Darwin and Francis Darwin were discovering that light and gravity were perceived by the tips of shoots and roots and that the stimulus was transmitted to other tissues, which underwent a growth response. These ideas were improved upon by Boysen-Jensen and Paál and were later developed into the Cholodny–Went hypothesis that tropisms were caused by the asymmetric distribution of a growth-promoting substance. These observations led to many efforts to identify this elusive growth-promoting substance, which we now know as auxin. In this review of auxin fi eld advances over the past century, we start with a seminal paper by Kenneth Thimann and Charles Schneider titled “The relative activities of different auxins” from the A merican Journal of Botany , in which they compare the growth altering properties of several auxinic compounds. From this point, we explore the modern molecular understanding of auxin—including its biosynthesis, transport, and perception. Finally, we end this review with a discussion of outstanding questions and future directions in the auxin fi eld. Over the past 100 yr, much of our progress in understanding auxin biology has relied on the steady and collective advance of the fi eld of auxin researchers; we expect that the next 100 yr of auxin research will likewise make many exciting advances. K ey wo rds: auxin; auxin history; metabolism; signaling; transport.E N D E R S A N D S T R A D E R—AU X I N AC T I V I T Y: PA S T, P R E S E N T, A N DF U T U R E •V O L. 102 ,N O. 2 F E B R UA RY 2015 •181activity were also discovered, either through chemical synthesis or extraction from biological sources. Structure–activity studies were beginning to emerge, and it was determined that a ring system containing a double bond as the nucleus of a molecule, a side chain, a carboxyl group, and a particular space relation-ship were necessary for cell elongation in the A vena test of auxin activity ( K oepfl i et al., 1938 ).T he burgeoning fi eld of auxin research was littered with op-posing opinions on the mechanism of auxin action and what compounds were considered auxins. However, the fi eld eventu-ally settled at least some of these disputes and auxin researchers W ent and Thimann (1937 , in P hytohormones, p. 5) provided insightful commentary on how such progress was made: “…in regard to the question of priority of the discovery of the auxins. We feel that the gradual unfolding of the current conceptions and the cooperation of different workers has made it impossible to credit any one person with such a discovery, and it is to be hoped that the reader … will gain the impression of a steady and collective advance rather than of individual contributions.”T HIMANN AND SCHNEIDER, 1939T himann and Schneider (1939) compared the activities of several compounds that had been recently identifi ed for auxin activity in the A vena assay. This comparison eventually led to structure–activity relationship studies by Thimann’s labora-tory and others that would reveal the defi ning characteristics of auxin molecules. The activity of indole-3-acetic acid (IAA), the most investigated auxin at the time of this study, was compared with the activity of six other auxin molecules: indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), indole-3-propionic acid (IPrA), phenyl-acetic acid (PAA or ФAA), phenyl-butyric acid (PBA or ФBA), and benzofurane-3-acetic acid (BzFA) ( F ig. 2 ).S tandard auxin assays at this time consisted of either incu-bating etiolated stem sections of A vena or P isum coleoptiles in auxin solutions and measuring coleoptile elongation or splitting etiolated P isum stems and measuring their degree of curvature after incubation in an auxin solution ( F ig. 1B ). In these assays, P isum was more sensitive to the auxins than A vena; however, the tested auxins displayed the same rela-tive activity in both species, with slightly different response curves. The major conclusion from Thimann and Schnei-der’s straight growth experiments was that both the absolute activities and the activities relative to IAA depended on the species of plant and that A vena and P isum did not respond to auxin in a similar way. These fi ndings were contrary to the belief at the time that species may respond to auxins differ-ently, but those differences would always be in the sameF ig. 1. Auxin response assays. (A) The A vena test. Compounds are tested for auxin activity in A vena sativa seedlings mounted on a test apparatus, and the tip of the coleoptile removed. After a few hours of growth, a larger portion of the coleoptile is removed, and the primary leaf pulled upward to detach it from the base.A block of agar containing the compound to be tested is placed on one side of the cut surface. After incubation, the curvature induced by the diffusion and transport of auxin from the agar block into the coleoptile of the A vena seedling is measured. Image modifi ed from W ent and Thimann (1937) . (B) The P isum test. Compounds are tested for auxin activity by removing the top of 7-d-old P isum sativum seedlings below the terminal bud and the stem split lengthwise for 3 cm. The split stem section is excised a few millimeters below the split and split stems incubated in solutions containing compounds to be tested. After incubation, the curvature angles induced from the auxins causing cell expansion on one side of the coleoptile are measured. Image modifi ed from W ent and Thimann (1937) . (C) The A rabidopsis root elongation test. Compounds are tested for auxin activity by plating sterilized seed of A rabidopsis thaliana on agar-solidifi ed media containing compounds of interest. After 7–10 d of growth, seedling root lengths are measured.182 • V O L. 102 ,N O. 2 F E B R UA RY 2015 • A M E R I C A N J O U R N A L O F B OTA N Yproportions. The idea that each species may have drastically different sensitivities to different auxins was fi rst demon-strated in this seminal paper.M any barriers limited early auxin research. For example, Thimann and Schneider recognized that differences in the pu-rity in their auxin stocks may have played a role in the differ-ing auxin activities they observed, a problem that plagued the fi eld at that time. Metabolism of the applied molecules into other compounds within the plant (for example, production of IAA conjugates and oxIAA, conversion of IBA to IAA) was diffi cult to track. Additionally, molecular genetic differences between species had not yet been uncovered, making under-standing of the response differences between A vena and P isum diffi cult.F ig. 2. Structures of naturally occurring and synthetic auxins. Auxins found in plants include the active auxins indole-3-acetic acid (IAA), 4-Cl-IAA, and phenyl-acetic acid (PAA), as well as the inactive auxin precursors indole-3-butyric acid (IBA), and indole-3-propionic acid (IPrA). Synthetic auxins include the active 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and the inactive precursor 2,4-dichlorophenoxybutyric acid (2,4-DB). The occurrence of auxin a(auxenotriolic acid), auxin b(auxenolonic acid), benzofurane-3-acetic acid (BzFA), and phenyl-butyric acid (PBA) in plants or other organisms has been debated or is unknown.E N D E R S A N D S T R A D E R—AU X I N AC T I V I T Y: PA S T, P R E S E N T, A N DF U T U R E •V O L. 102 ,N O. 2 F E B R UA RY 2015 •183A UXIN RESEARCH AFTER WENT,THIMANN,AND SCHNEIDERP hytohormones( W ent and Thimann, 1937 ) lays out an ex-cellent snapshot of the knowledge of the auxin fi eld in the late 1930s. However, many important discoveries were made be-tween 1939 and the molecular revolution. Until the 1980s, auxin researchers were limited by the techniques of the time and were restricted to mostly investigating the physiological effects of auxin application. However, as more sophisticated techniques were developed, more mechanistic observations were made, such as the characterization of a two-phase growth response to auxin, the characterization of auxin polar transport, and the contributions of transcription and protein synthesis to auxin re-sponse (reviewed by G oldsmith, 1977 ;K ey, 1989 ;T himann, 1977 ). These larger-picture characterizations laid the ground-work for the molecular revolution and provided clues as to what categories of genes could be involved in auxin response.M ODERN-DAY MOLECULAR UNDERSTANDING OFAUXIN ACTIONO ur molecular understanding of auxin activity has exploded over the past 20 yr. After the establishment of A rabidopsis as a model organism for plant molecular biology, it quickly became a target of auxin research. The modern A rabidopsis equivalent to the classic A vena( F ig. 1A ) and P isum split stem ( F ig. 1B ) tests is the measurement of auxin inhibition of root elongation in A rabidopsis( F ig. 1C ), based on the inhibitory properties of auxin on root elongation (reviewed by S cott, 1972 ). Use of A ra-bidopsis mutant screens based on resistance to the inhibitory effects of auxin on root elongation uncovered many of the fac-tors required for the three main modes of regulation: metabo-lism, transport, and signaling. In the following three sections, we summarize our current understanding of these three modes of regulation of auxin activity (metabolism, transport, and sig-naling) and outline possible areas of future studies to gain a deeper understanding of auxin biology.A UXIN METABOLISMT he natural auxin indole-3-acetic acid (IAA) is synthesized through both tryptophan (Trp)-dependent and Trp-independent pathways (reviewed by K orasick et al., 2013 ;T ivendale et al., 2014 ). Five major naturally occurring compounds are recog-nized as IAA precursors, including indole-3-pyruvic acid (IPyA), indole-3-acetaldoxime (IAOx), indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), and indole-3-acetaldehyde (IAAld) ( F ig. 3A ). The IPyA pathway is considered the “main” auxin biosynthetic pathway (reviewed by Z hao, 2012 ). A rabi-dopsis responds differently to these auxin precursors, display-ing the greatest sensitivity to IPyA and the least sensitivity to IAM in root elongation assays ( F ig. 3B ).A uxin was hypothesized to be derived from tryptophan as early as the 1930s (reviewed by W ent and Thimann, 1937 ). However, the major mechanism of conversion from tryptophan to IAA remained elusive until relatively recently. The IPyA pathway appears to be the main contributor to IAA (reviewed by K orasick et al., 2013 ;T ivendale et al., 2014 ) and is the only completely described pathway to date. The TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) family of enzymes converts tryptophan to IPyA ( S tepanova et al., 2008 ;T ao et al., 2008 ;Y amada et al., 2009 ), and the YUCCA (YUC) family of enzymes converts IPyA to IAA ( M ashiguchi et al., 2011 ;S tepanova et al., 2011 ;W on et al., 2011 ;D ai et al., 2013 ), creating a simple, two-step conversion of Trp to IAA. The discovery of this two-step IAA biosynthesis pathway caused great excitement within the auxin community because it simplifi ed the possible models for auxin biosynthesis ( F ig. 3A ). T he IAOx pathway may be restricted to the Brassicaceae ( Q uittenden et al., 2009 ;S ugawara et al., 2009 ). In A rabidopsis, the cytochrome P450 enzymes CYP79B2 and CYP79B3 con-vert tryptophan to IAOx ( H ull et al., 2000 ;M ikkelsen et al., 2000 ;Z hao et al., 2002 ). IAOx is largely used for production of indole glucosinolate defense compounds (B ak et al., 2001 ; Z hao et al., 2002 ;M ikkelsen et al., 2004 ), with only a small portion converted to IAA ( Z hao et al., 2002 ;S ugawara et al., 2009 ). A minor role for the IAOx pathway in IAA production has been suggested because the c yp79b2 cyp79b3double mu-tant has little effect on IAA levels ( L jung et al., 2005 ;S ugawara et al., 2009 ). However, hyperaccumulation of IAOx in the s u-perroot1and s uperroot2mutants results in greatly increased levels of IAA ( M ikkelsen et al., 2004 ), suggesting that, at least under some conditions, IAOx has the potential of substantially contributing to the pool of IAA. The c yp79b2 cyp79b3double mutant displays decreased IAN ( Z hao et al., 2002 ;S ugawara et al., 2009 ) and IAM ( S ugawara et al., 2009 ) levels, suggesting IAOx can be converted to both IAN and IAM. The enzymatic steps between IAOx and IAN remain unknown at this time; however, it appears that IAN can be converted to IAA through the activity of nitrolases ( S chmidt et al., 1996 ;N ormanly et al., 1997 ).I ndole-3-acetamide (IAM) has been identifi ed as an endoge-nous auxin precursor in many species throughout the plant king-dom (reviewed by K orasick et al., 2013 ). In A rabidopsis,IAM may be produced from IAOx ( S ugawara et al., 2009 ). Intrigu-ingly, IAM has been detected in many species in which IAOx has not been detected (reviewed by K orasick et al., 2013 ), sug-gesting that IAM can be produced from precursors other than IAOx ( S ugawara et al., 2009 ). Conversion of IAM to IAA may be facilitated by AMIDASE1 (AMI1) ( P ollmann et al., 2003 ). O ur understanding of tryptophan-dependent de novo auxin biosynthesis has been greatly improved by the discovery of the two-step IPyA pathway. However, much less attention has been paid to the tryptophan-independent pathways of IAA biosyn-thesis. For reviews of Trp-independent auxin biosynthesis, please see K orasick et al. (2013) and T ivendale et al. (2014) .I n addition to de novo IAA biosynthesis, many inactive IAA storage forms, including IAA conjugates, methyl ester IAA (MeIAA), and IBA can be rapidly converted to free IAA to regulate auxin homeostasis (reviewed by K orasick et al., 2013 ). These auxin storage forms are postulated to infl uence auxin sensitivity, transport, and compartmentalization ( C ohen and Bandurski, 1982 ). Although these storage forms can accumu-late to high levels in the plant ( T able 1 ), specifi c roles for auxin derived from these inactive molecules are only now beginning to be understood. For example, altering IBA-to-IAA conver-sion results in an array of seedling developmental defects, in-cluding defects in cotyledon and root hair cell expansion, apical hook curvature, high-temperature-induced hypocotyl elongation, lateral root production, and root meristem maintenance, with-out obvious effects on adult plant morphology ( S trader et al., 2010 ,2011 ) whereas altering IBA conjugation to glucose alters shoot branching and drought tolerance in adult plant tissues184 • V O L. 102 ,N O. 2 F E B R UA RY 2015 • A M E R I C A N J O U R N A L O F B OTA N YF ig. 3. Auxin biosynthesis and storage forms in higher plants. (A) Possible pathways for plant auxin biosynthesis. Solid arrows indicate those steps for which enzymes are known. Dashed arrows indicate those steps for which no enzyme has been identifi ed or the enzyme identity is in question. (B) Mean primary root lengths ( ±S E) of 8-d-old A rabidopsis seedlings grown in the presence of the indicated auxin or auxin precursor.E N D E R S A N D S T R A D E R—AU X I N AC T I V I T Y: PA S T, P R E S E N T, A N DF U T U R E •V O L. 102 ,N O. 2 F E B R UA RY 2015 •185T ABLE1. IAA and IAA precursor levels in A rabidopsis thaliana. Molecule Level (ng/gfw)SourceIAOx 1.7S ugawara et al., 2009 IAN9720.0S ugawara et al., 2009 IAM9.9S ugawara et al., 2009 IPyA56.0M ashiguchi et al., 2011 IAAld11.3M ashiguchi et al., 2011 IBA 1.3S trader et al., 2010IAA-Asp17.4T am et al., 2000IAA-Glu 3.5T am et al., 2000IAA-Glc12.0T am et al., 2000IAA11.2S ugawara et al., 2009( T ognetti et al., 2010 ). In addition, blocking conversion of IAA-amino acid conjugates to free IAA results in decreased lateral root production ( R ampey et al., 2004 ), decreased hypo-cotyl elongation ( R ampey et al., 2004 ), and decreased root hair elongation ( S trader et al., 2010 ), suggesting that conjugate-derived auxin plays roles in these developmental processes. Al-tering IAA conversion to MeIAA by either altering expression of I AA CAROXYMETHYLTRANSFERASE1(IAMT1) or mutat-ing M ETHYL ESTERASE17(MES17), results in phenotypes that suggest that MeIAA-derived auxin plays roles in gravitrop-ism, leaf epinasty, plant height, and fertility ( Q in et al., 2005 ; Y ang et al., 2008 ). Intriguingly, blocking conversion of multi-ple auxin storage forms to free IAA results in compensating increased activity of the IPyA pathway ( S piess et al., 2014 ). Specific roles for auxin derived from these storage forms is only now being appreciated, and more work will be necessary to understand the conditions under which plants use de novo auxin biosynthesis vs. conversion of storage forms to increase free auxin levels.I n addition to IAA, 4-Cl-IAA and phenylacetic acid (PAA) are active, naturally occurring auxins (reviewed by K orasick et al., 2013 ). In legumes, 4-Cl-IAA appears to be the major ac-tive auxin, whereas PAA occurrence is more widespread. In-triguingly, both 4-Cl-IAA and PAA appear to be synthesized by the TAA1 and YUCCA families of enzymes from 4-Cl-Trp and phenylalanine, respectively ( T ivendale et al., 2012 ;D ai et al., 2013 ). The importance of these auxin molecules, which are present at physiologically relevant levels, is starting to be ap-preciated (see S trader and Nemhauser, 2013 ), although much research is still necessary to understand specifi c roles for these active auxin molecules in the plant.A uxin metabolism has had a complicated history ( T ivendale et al., 2014 ). As we learn more about how auxin is synthesized, stored, and degraded, we will come closer to a more complete understanding of how these pathways integrate to facilitate auxin-regulated growth responses. For example, do certain auxin biosynthetic pathways contribute more strongly to auxin homeo-stasis than the IPyA pathway under specifi c environmental or developmental contexts? Comparing differences in auxin me-tabolism in different plant species will surely help us reach this goal. Uncovering the biological signifi cance of these pathways will undoubtedly be a challenging goal for the coming century.A UXIN TRANSPORTL ong before the chemical identity of auxin was known, the rapid and polar transport of auxin was known to be important to growth responses. In P hytohormones( 1937), Went and Thimann speculated that auxin is not transported by simple diffusion, but by some other mechanism. We now know that specialized car-riers mediate auxin transport. Members of the PIN-FORMED (PIN) and ATP-BINDING CASSETTE SUBFAMILY B (ABCB) protein families facilitate the effl ux of IAA, whereas members of the AUXIN RESISTANT1/LIKE AUX1 (AUX1/ LAX) transporters facilitate IAA uptake ( F ig. 4 ;reviewed by Z azimalova et al., 2010 ;P eer et al., 2011 ).A UX1/LAX family members, which resemble amino acid permeases, include the plasma-membrane localized H +sym-porters AUX1, LAX1, LAX2, and LAX3. AUX1 facilitates up-take of both the natural auxin IAA and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) ( M aher and Martindale, 1980 ;Y amamoto and Yamamoto, 1998 ;M archant et al., 1999 ; S warup et al., 2004 ;Y ang et al., 2006 ), but not the synthetic auxin NAA ( Y amamoto and Yamamoto, 1998 ;M archant et al., 1999 ;Y ang et al., 2006 ) or the natural auxin precursor IBA (reviewed by S trader and Bartel, 2011 ). LAX1 ( Péret et al., 2012 ) and LAX3 ( S warup et al., 2008 ) are infl ux transporters of the natural auxin IAA and the synthetic auxin 2,4-D, but not the synthetic auxin NAA. The transport specifi cities and locations of AUX1 family members may contribute to auxin response differences.T he diversity of phenotypes displayed by mutants defective in members of the AUX/LAX family reveal distinct roles forF ig. 4. Auxin transport mechanisms. AUX/LAX proteins localize to different faces of the cell depending on the particular cell type, where they act to infl ux auxin from the apoplast into the cytoplasm (depicted in green). The long PIN proteins localize to either the apical or basal face of the cell in the root to effl ux auxin and establish auxin gradients. The short PIN proteins and PILS proteins localize to the ER, where they effl ux auxin from the cytoplasm into the ER lumen, presumably to regulate auxin activity via compartmentalization, and possibly metabolism. The ABCB family of auxin transporters localize to the plasma membrane to effl ux auxin outside the cell. Some members of the ABCB family have been shown to display both effl ux and infl ux activity based on the cyto-plasmic concentration of auxin. Two members of the ABCG family localize to the outer lateral domain in the epidermis, and transport IBA into the surround-ing environment.186 • V O L. 102 ,N O. 2 F E B R UA RY 2015 • A M E R I C A N J O U R N A L O F B OTA N Ythese auxin carriers. The activity of AUX1 is necessary for plant gravitropic responses ( B ennett et al., 1996 ;M archant et al., 1999 ), root hair development ( G rebe et al., 2002 ;J ones et al., 2009 ), apical hook development ( V andenbussche et al., 2010 ), and phyllotaxis ( R einhardt et al., 2003 ;B ainbridge et al., 2008 ). The l ax3mutant seedlings display decreased lateral root emer-gence, increased numbers of lateral root primordia, and de-creased expression of cell-wall-modifying enzymes ( S warup et al., 2008 ), suggesting that LAX3 plays roles in auxin-dependent cell wall modification for cortical cell separation and lateral root emergence. Additionally, LAX3 is involved in apical hook development ( V andenbussche et al., 2010 ). LAX1 and LAX2 regulate phyllotaxis ( B ainbridge et al., 2008 ), and LAX2 also regulates vascular differentiation in cotyledons ( Péret et al., 2012 ). These auxin infl ux carriers are critical for polar auxin trans-port and play roles in diverse aspects of plant development.P IN and ABCB family members mediate auxin effl ux from the cell. PIN proteins are gradient-driven auxin effl ux carriers that are unique to plants. There are eight members of the PIN family in A rabidopsis, which are divided into “long” and “short” PIN categories. Long PIN proteins include PIN1, PIN2, PIN3, PIN4, and PIN7, which polarly localize to the plasma membrane (reviewed in Z azimalova et al., 2010 ). Direct trans-port of IAA has been shown for the plasma-membrane-local-ized PIN1 ( P etrášek et al., 2006 ;Y ang and Murphy, 2009 ), PIN2 ( C hen et al., 1998 ;P etrášek et al., 2006 ;Y ang and Murphy, 2009 ), PIN3 ( F riml et al., 2002a ), PIN4 ( F riml et al., 2002b ), and PIN7 ( P etrášek et al., 2006 ;Y ang and Murphy, 2009 ). Intriguingly, each of these PIN proteins displays specifi c and distinct localization. In root tissues, PIN1 is localized to the downward face of stele, endodermal, and pericycle cells ( F riml et al., 2002b ;B lakeslee et al., 2007 ). PIN2 is localized in corti-cal and epidermal cells ( Müller et al., 1998 ;B lakeslee et al., 2007 ) and the lateral root cap ( F riml, 2003 ), whereas PIN3 is present throughout the root pericycle and columella ( F riml et al., 2002a ). PIN4 localizes to the root meristem ( F riml et al., 2002b ), and PIN7 is present in the columella and stele ( B lilou et al., 2005 ). The specifi c expression patterns and polar localization of these PIN proteins are necessary to establish differential dis-tribution of auxin to regulate aspects of plant development (re-viewed by Z azimalova et al., 2010 ).A lthough all examined PIN proteins transport IAA, PIN fam-ily members differ in their ability to transport other auxinic compounds. For example, PIN2 and PIN7 effl ux 2,4-D ( Y ang and Murphy, 2009 ), whereas PIN1 does not display this ability ( Y ang and Murphy, 2009 ). Additionally, PIN4 and PIN7 trans-port NAA ( P etrášek et al., 2006 ), whereas PIN1 and PIN2 do not (B lakeslee et al., 2007 ). These differing substrate specifi ci-ties may contribute to differences in whole-plant responses to varying auxinic compounds.U nlike the “long” PIN proteins involved in polar transport of IAA, the “short” PIN proteins, PIN5 ( M ravec et al., 2009 ;G anguly et al., 2010 ;D ing et al., 2012 ), PIN6 ( M ravec et al., 2009 ;S awchuk et al., 2013 ), and PIN8 ( M ravec et al., 2009 ;G anguly et al., 2010 ;D al Bosco et al., 2012 ;D ing et al., 2012 ; S awchuk et al., 2013 ) localize to the endoplasmic reticulum (ER) to transport IAA from the cytoplasm into the ER ( M ravec et al., 2009 ;D al Bosco et al., 2012 ;D ing et al., 2012 ;S awchuk et al., 2013 ). PIN5, PIN6, and PIN8 additionally transport NAA ( P etrášek et al., 2006 ;M ravec et al., 2009 ;G anguly et al., 2010 ). At this time, specifi c roles for transport of auxin into the ER to alter plant growth are unknown; however, it seems likely that this may serve to alter cytoplasmic levels of free IAA. In addition to PIN5, PIN6, and PIN8, the PIN-LIKES (PILS) also facilitate auxin infl ux into the ER and may have roles in regulat-ing the cytoplasmic pool of free auxin and auxin–amino acid conjugates ( B arbez et al., 2012 ). Future research may reveal exciting roles for these ER-localized transporters in regulating auxin levels under certain conditions.S tudy of the polar localization of PIN proteins has been in-strumental in understanding plant cell polarity. The polar lo-calization of these PIN transporters requires endocytosis and selective recycling back to the plasma membrane ( G eldner et al., 2001 ;K leine-Vehn and Friml, 2008 ). Recycling of PIN proteins presumably allows for rapid relocalization in re-sponse to environmental or developmental cues, such as gra-vitropism or embryonic development (reviewed by L ofke et al., 2013 ).I n addition to PIN-mediated IAA efflux, several ABCB transporters are required for IAA efflux, including ABCB1, ABCB4, ABCB19, and ABCB21 (reviewed by R emy and Duque, 2014 ). Both ABCB1 and ABCB19 facilitate the effl ux of IAA ( N oh et al., 2001 ;G eisler et al., 2005 ;L ewis et al., 2007 ) and 2,4-D ( Y ang and Murphy, 2009 ). ABCB4 functions in the effl ux of IAA ( S antelia et al., 2005 ) and NAA ( C ho et al., 2007 ) and localizes to the plasma membrane and endomem-brane compartments of the root ( T erasaka et al., 2005 ;C ho et al., 2007 ). Intriguingly, ABCB4 and ABCB21 may switch from influx to efflux functions, depending on internal auxin concentrations, importing auxin at low cytoplasmic concentra-tions, and exporting auxin at higher concentrations ( Y ang and Murphy, 2009 ;K amimoto et al., 2012 ;K ubes et al., 2012 ). Structural differences in ABCB4 and ABCB21 from other ABCB proteins may allow for this unique activity ( Y ang and Murphy, 2009 ;K amimoto et al., 2012 ;K ubes et al., 2012 ). In addition, ABCB14 and ABCB15 are required for polar auxin transport ( K aneda et al., 2011 ), although it is unknown whether auxin is a substrate for these transporters. Phenotypes of mu-tants defective in ABCB family members suggest that these auxin transporters play key roles in plant development (re-viewed in P etrášek and Friml, 2009 ;P eer et al., 2011 ).I n addition to transport of active auxins (i.e., IAA, 2,4-D, and NAA), plants also transport the endogenous auxin precursor IBA (reviewed by S trader and Bartel, 2011 ;M ichniewicz et al., 2014 ). Early IBA transport assays relied on the ability of IBA to affect plant morphology distant from the site of application ( W ent and White, 1939 ;L eopold and Lam, 1961 ;Y ang and Davies, 1999 ), raising the question of whether IBA itself or IBA-derived IAA moved through these tissues to create these morphological changes. Later studies using radiolabeled or heavy IBA demonstrated that IBA and/or IBA conjugates may travel long distances through plant tissues ( L udwig-Müller et al., 1995a ;R ashotte et al., 2003 ;P oupart et al., 2005 ;L iu et al., 2012 ). Because IAA and IBA are chemically similar, one might hypothesize that these compounds are transported by the same mechanism. However, examined IAA carriers, including AUX1, PIN2, PIN7, ABCB1, and ABCB19, do not transport IBA (re-viewed in S trader and Bartel, 2011 ), suggesting that unique car-riers are required for IBA transport.S everal members of the A rabidopsis ABCG family of trans-porters are required for IBA, but not IAA, transport (reviewed in M ichniewicz et al., 2014 ). ABCG36 is likely an effl ux trans-porter of IBA but not IAA ( S trader and Bartel, 2009 ;Růžička et al., 2010 ), whereas ABCG37 is an effl ux carrier of IBA and 2,4-D but not IAA ( I to and Gray, 2006 ;S trader et al., 2008b ; Růžička et al., 2010 ). Intriguingly, ABCG36 and ABCG37 are。