New insight into the biochemical mechanisms regulating auxin transport in plants

The transport of the plant hormone auxin has been under intense investigation since its identification 80 years ago. Studies have gradually refined our understanding of the importance of auxin transport in many aspects of plant signalling and development, and the focus has intensified in recent years towards the identification of the proteins involved in auxin transport and their functional mechanism. Within the past 18 months, the field has progressed rapidly, with confirmation that several distinct classes of proteins, previously dubbed as 'putative auxin permeases' or 'auxin transport facilitators', are bona fide transporters of IAA (indol-3-ylacetic acid). In this review we will appraise the recent transport data and highlight likely future research directions, including the characterization of auxiliary proteins necessary for the regulation of auxin transporters.     

Specificity and similarity of functions of the Aux/IAA genes in auxin signaling of Arabidopsis revealed by promoter-exchange experiments among MSG2/IAA19, AXR2/IAA7, and SLR/IAA14

As indicated by various and some overlapped phenotypes of the dominant mutants, the Aux/IAA genes of Arabidopsis (Arabidopsis thaliana) concomitantly exhibit a functional similarity and differentiation. To evaluate the contributions of their expression patterns determined by promoter activity and molecular properties of their gene products to Aux/IAA function, we examined phenotypes of transgenic plants expressing the green fluorescent protein (GFP)-tagged msg2-1/iaa19, axr2-1/iaa7, or slr-1/iaa14 cDNA by the MSG2 or AXR2 promoter. When driven by the MSG2 promoter (pMSG2), each GFP-tagged cDNA caused the msg2-1 phenotype, that is, the wild-type stature in the mature-plant stage, long and straight hypocotyls in the dark, reduced lateral root formation, relatively mild agravitropic traits in hypocotyls, and a normal gravitropic response in roots. However, development of one or two cotyledonary primordia was often arrested in embryogenesis of the pMSG2::axr2-1::GFP and pMSG2::slr-1::GFP plants, resulting in monocotyledonary or no cotyledonary seedlings. Such defects in embryogenesis were never seen in pMSG2::msg2-1::GFP or the msg2-1, axr2-1, or slr-1 mutant. The MSG2 promoter-GUS staining showed that expression of MSG2 started specifically in cotyledonary primordia of the triangular-stage embryos. When driven by the AXR2 promoter (pAXR2), each GFP-tagged mutant cDNA caused, in principle, aberrant aboveground phenotypes of the corresponding dominant mutant. However, either the axr2-1::GFP or slr-1::GFP cDNA brought about dwarf, agravitropic stems almost identical to those of axr2-1, and the pAXR2::msg2-1::GFP and pAXR2::slr-1::GFP hypocotyls exhibited complete loss of gravitropism as did axr2-1. These results showed functional differences among the msg2-1, axr2-1, and slr-1 proteins, though some phenotypes were determined by the promoter activity.     

AXR2 encodes a member of the Aux/IAA protein family

The dominant gain-of-function axr2-1 mutation of Arabidopsis causes agravitropic root and shoot growth, a short hypocotyl and stem, and auxin-resistant root growth. We have cloned the AXR2 gene using a map-based approach, and find that it is the same as IAA7, a member of the IAA (indole-3-acetic acid) family of auxin-inducible genes. The axr2-1 mutation changes a single amino acid in conserved domain II of AXR2/IAA7. We isolated loss-of-function mutations in AXR2/IAA7 as intragenic suppressors of axr2-1 or in a screen for insertion mutations in IAA genes. A null mutant has a slightly longer hypocotyl than wild-type plants, indicating that AXR2/IAA7 controls development in light-grown seedlings, perhaps in concert with other gene products. Dark-grown axr2-1 mutant plants have short hypocotyls and make leaves, suggesting that activation of AXR2/IAA7 is sufficient to induce morphological responses normally elicited by light. Previously described semidominant mutations in two other Arabidopsis IAA genes cause some of the same phenotypes as axr2-1, but also cause distinct phenotypes. These results illustrate functional differences among members of the Arabidopsis IAA gene family.     

Dynamics of IAA and cytokinins in flower tissues of transgenic tobacco mutant plants with mutant phenotype

A comparative analysis of IAA and total cytokinin contents at various developmental stages of floral morphogenesis (I–XII) in transgenic tobacco (Nicotiana tabacum L.) plants with mutant phenotypes was performed. Disturbances in flower structure were correlated with changes in the content and dynamics of these phytohormones. In plants with the phenotype “wavy corolla”, IAA content increased by three times as compared with control at stage VIII; in plants with the phenotype “longostyly”, IAA content increased gradually from VIII to XII stage and sharply (by nine times) at stage XII. In plants with the phenotype “wavy corolla”, total content of cytokinins was lower than in control at all developmental stages (except for stage I), whereas their dynamics coincided with that in control flowers. In plants with the phenotype “longostyly”, cytokinin content was twice lower than in control at early developmental stages, whereas it oscillated at later stages, approaching to control value at stage XII. It is supposed that differences observed could result from T-DNA insertion into plant own genes, which led to changes in the content and dynamics of phytohormones in developing flowers and production of anomalies in the flower structure.     

Arabidopsis AXR6 encodes CUL1 implicating SCF E3 ligases in auxin regulation of embryogenesis

The AXR6 gene is required for auxin signaling in the Arabidopsis embryo and during postembryonic development. One of the effects of auxin is to stimulate degradation of the Aux/IAA auxin response proteins through the action of the ubiquitin protein ligase SCF(TIR1). Here we show that AXR6 encodes the SCF subunit CUL1. The axr6 mutations affect the ability of mutant CUL1 to assemble into stable SCF complexes resulting in reduced degradation of the SCF(TIR1) substrate AXR2/IAA7. In addition, we show that CUL1 is required for lateral organ initiation in the shoot apical meristem and the inflorescence meristem. These results indicate that the embryonic axr6 phenotype is related to a defect in SCF function and accumulation of Aux/IAA proteins such as BDL/IAA12. In addition, we show that CUL1 has a role in auxin response throughout the life cycle of the plant.     

Multiple AUX/IAA-ARF modules regulate lateral root formation: the role of Arabidopsis SHY2/IAA3-mediated auxin signalling

In Arabidopsis thaliana, lateral root (LR) formation is regulated by multiple auxin/indole-3-acetic acid (Aux/IAA)-AUXIN RESPONSE FACTOR (ARF) modules: (i) the IAA28-ARFs module regulates LR founder cell specification; (ii) the SOLITARY-ROOT (SLR)/IAA14-ARF7-ARF19 module regulates nuclear migration and asymmetric cell divisions of the LR founder cells for LR initiation; and (iii) the BODENLOS/IAA12-MONOPTEROS/ARF5 module also regulates LR initiation and organogenesis. The number of Aux/IAA-ARF modules involved in LR formation remains unknown. In this study, we isolated the shy2-101 mutant, a gain-of-function allele of short hypocotyl2/suppressor of hy2 (shy2)/iaa3 in the Columbia accession. We demonstrated that the shy2-101 mutation not only strongly inhibits LR primordium development and emergence but also significantly increases the number of LR initiation sites with the activation of LATERAL ORGAN BOUNDARIES-DOMAIN16/ASYMMETRIC LEAVES2-LIKE18, a target gene of the SLR/IAA14-ARF7-ARF19 module. Genetic analysis revealed that enhanced LR initiation in shy2-101 depended on the SLR/IAA14-ARF7-ARF19 module. We also showed that the shy2 roots contain higher levels of endogenous IAA. These observations indicate that the SHY2/IAA3-ARF-signalling module regulates not only LR primordium development and emergence after SLR/IAA14-ARF7-ARF19 module-dependent LR initiation but also inhibits LR initiation by affecting auxin homeostasis, suggesting that multiple Aux/IAA-ARF modules cooperatively regulate the developmental steps during LR formation.     

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.     

The plant hormone auxin: insight in sight.

Physiological experiments conducted over the last 60 years indicate that the plant hormone auxin regulates a diverse set of developmental processes via changes in cell division, cell elongation and cell differentiation. Recent studies using transgenic plants with altered auxin levels support these conclusions and promise to provide more detailed information on the role of auxin during plant development. Although it is possible that all auxin responses are mediated by the same primary biochemical events, the studies described in this review are more consistent with multiple modes of auxin action. The development of molecular and genetic approaches to the study of hormone action should resolve this issue. The accelerated rate of progress in this field suggests that real insight into the mechanism of auxin action may be forthcoming.     

Proinflammatory cytokines: an insight into pancreatic oncogenesis

Pancreatic cancer is a highly lethal disease, being one of the five leading death causes among oncologic patients. It is usually diagnosed late due to the paucity of clinical signs, and the current therapy means have limited success. One of the documented risk factors for developing pancreatic adenocarcinoma is chronic pancreatitis. It is postulated that a chronic inflammatory disease has a potential of evolving toward neoplasia, a fact that could account for a percentage of the pancreatic cancers. Starting from this assumption, we intended to analyze the serum reflection of some molecules with proinflammatory roles, and compare them in healthy individuals, in patients with chronic pancreatitis and with pancreatic adenocarcinoma. Additionally, we performed a biochemical and hematological assessment of the study groups, and compared the results with the immunological parameters analyzed in the same subjects. We found significantly higher levels of Tumor Necrosis Factor-alpha and Interleukin 6 in chronic pancreatitis and pancreatic adenocarcinoma sera (with higher levels in the pancreatitis group than in the cancer group), compared to healthy controls. Additionally, we found significantly higher levels of interleukin 8 and Macrophage Inflammatory Protein-3 alpha in pancreatic cancer, compared to chronic pancreatitis and controls. We also identified numerous correlations between the abovementioned cytokines/chemokines and biochemical parameters, not very much studied before. Our results plead for a pathogenic role of chronic inflammation in pancreatic carcinogenesis, thus offering a potential tool for earliy diagnose or targets for therapy.     

Double agent indole-3-acetic acid: mechanistic analysis of indole-3-acetaldehyde dehydrogenase AldA that synthesizes IAA,an auxin that aids bacterial virulence

The large diversity of organisms inhabiting various environmental niches on our planet are engaged in a lively exchange of biomolecules, including nutrients, hormones, and vitamins. In a quest to survive, organisms that we define as pathogens employ innovative methods to extract valuable resources from their host leading to an infection. One such instance is where plant-associated bacterial pathogens synthesize and deploy hormones or their molecular mimics to manipulate the physiology of the host plant. This commentary describes one such specific example—the mechanism of the enzyme AldA, an aldehyde dehydrogenase (ALDH) from the bacterial plant pathogen Pseudomonas syringae which produces the plant auxin hormone indole-3-acetic acid (IAA) by oxidizing the substrate indole-3-acetaldehyde (IAAld) using the cofactor nicotinamide adenine dinucleotide (NAD⁺) (Bioscience Reports (2020) 40(12), https://doi.org/10.1042/BSR20202959). Using mutagenesis, enzyme kinetics, and structural analysis, Zhang et al. established that the progress of the reaction hinges on the formation of two distinct conformations of NAD(H) during the reaction course. Additionally, a key mutation in the AldA active site ‘aromatic box’ changes the enzyme’s preference for an aromatic substrate to an aliphatic one. Our commentary concludes that such molecular level investigations help to establish the nature of the dynamics of NAD(H) in ALDH-catalyzed reactions, and further show that the key active site residues control substrate specificity. We also contemplate that insights from the present study can be used to engineer novel ALDH enzymes for environmental, health, and industrial applications.     

The Arabidopsis Aux/IAA protein family has diversified in degradation and auxin responsiveness

Rapid, auxin-responsive degradation of multiple auxin/indole-3-acetic acid (Aux/IAA) proteins is essential for plant growth and development. Domain II residues were previously shown to be required for the degradation of several Arabidopsis thaliana Aux/IAA proteins. We examined the degradation of additional full-length family members and the proteolytic importance of N-terminal residues outside domain II using luciferase (LUC) fusions. Elimination of domain I did not affect degradation. However, substituting an Arg for a conserved Lys between domains I and II specifically impaired basal degradation without compromising the auxin-mediated acceleration of degradation. IAA8, IAA9, and IAA28 contain domain II and a conserved Lys, but they were degraded more slowly than previously characterized family members when expressed as LUC fusions, suggesting that sequences outside domain II influence proteolysis. We analyzed the degradation of IAA31, with a region somewhat similar to domain II but without the conserved Lys, and of IAA20, which lacks domain II and the conserved Lys. Both IAA20:LUC and epitope-tagged IAA20 were long-lived, and their longevity was not influenced by auxin. Epitope-tagged IAA31 was long-lived, like IAA20, but by contrast, it showed accelerated degradation in response to auxin. The existence of long-lived and auxin-insensitive Aux/IAA proteins suggeststhat they may play a novel role in auxin signaling.