Identification of IAA-regulated genes in Pseudomonas syringae pv. tomato strain DC3000

The auxin indole-3-acetic acid (IAA) is a plant hormone that not only regulates plant growth and development but also plays important roles in plant-microbe interactions. We previously reported that IAA alters expression of several virulence-related genes in the plant pathogen Pseudomonas syringae pv. tomato strain DC3000 ( Pto DC3000). To learn more about the impact of IAA on regulation of Pto DC3000 gene expression we performed a global transcriptomic analysis of bacteria grown in culture, in the presence or absence of exogenous IAA. We observed that IAA repressed expression of genes involved in the Type III secretion (T3S) system and motility and promoted expression of several known and putative transcriptional regulators. Several of these regulators are orthologs of factors known to regulate stress responses and accordingly expression of several stress response-related genes was also upregulated by IAA. Similar trends in expression for several genes were also observed by RT-qPCR. Using an Arabidopsis thaliana auxin receptor mutant that accumulates elevated auxin, we found that many of the P. syringae genes regulated by IAA in vitro were also regulated by auxin in planta . Collectively the data indicate that IAA modulates many aspects of Pto DC3000 biology, presumably to promote both virulence and survival under stressful conditions, including those encountered in or on plant leaves. IMPORTANCE Indole-3-acetic acid (IAA), a form of the plant hormone auxin, is used by many plant-associated bacteria as a cue to sense the plant environment. Previously, we showed that IAA can promote disease in interactions between the plant pathogen Pseudomonas syringae strain Pto DC000 and one of its hosts, Arabidopsis thaliana . However, the mechanisms by which IAA impacts the biology of Pto DC3000 and promotes disease are not well understood. Here we demonstrate that IAA is a signal molecule that regulates gene expression in Pto DC3000. The presence of exogenous IAA affects expression of over 700 genes in the bacteria, including genes involved in Type III secretion and genes involved in stress response. This work offers insight into the roles of auxin promoting pathogenesis.

Key auxin response factor (ARF) genes constraining wheat tillering of mutant dmc

Tillering ability is a key agronomy trait for wheat (Triticum aestivum L.) production. Studies on a dwarf monoculm wheat mutant (dmc) showed that ARF11 played an important role in tillering of wheat. In this study, a total of 67 ARF family members were identified and clustered to two main classes with four subgroups based on their protein structures. The promoter regions of T. aestivum ARF (TaARF) genes contain a large number of cis-acting elements closely related to plant growth and development, and hormone response. The segmental duplication events occurred commonly and played a major role in the expansion of TaARFs. The gene collinearity degrees of the ARFs between wheat and other grasses, rice and maize, were significantly high. The evolution distances among TaARFs determine their expression profiles, such as homoeologous genes have similar expression profiles, like TaARF4-3A-1, TaARF4-3A-2 and their homoeologous genes. The expression profiles of TaARFs in various tissues or organs indicated TaARF3, TaARF4, TaARF9 and TaARF22 and their homoeologous genes played basic roles during wheat development. TaARF4, TaARF9, TaARF12, TaARF15, TaARF17, TaARF21, TaARF25 and their homoeologous genes probably played basic roles in tiller development. qRT-PCR analyses of 20 representative TaARF genes revealed that the abnormal expressions of TaARF11 and TaARF14 were major causes constraining the tillering of dmc. Indole-3-acetic acid (IAA) contents in dmc were significantly less than that in Guomai 301 at key tillering stages. Exogenous IAA application significantly promoted wheat tillering, and affected the transcriptions of TaARFs. These data suggested that TaARFs as well as IAA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of ARFs in wheat.

Overexpression of SgGH3.1 from Fine-Stem Stylo (Stylosanthes guianensis var. intermedia) Enhances Chilling and Cold Tolerance in Arabidopsis thaliana

Stylosanthes (stylo) species are commercially significant tropical and subtropical forage and pasture legumes that are vulnerable to chilling and frost. However, little is known about the molecular mechanisms behind stylos’ responses to low temperature stress. Gretchen-Hagen 3 (GH3) proteins have been extensively investigated in many plant species for their roles in auxin homeostasis and abiotic stress responses, but none have been reported in stylos. SgGH3.1, a cold-responsive gene identified in a whole transcriptome profiling study of fine-stem stylo (S. guianensis var. intermedia) was further investigated for its involvement in cold stress tolerance. SgGH3.1 shared a high percentage of identity with 14 leguminous GH3 proteins, ranging from 79% to 93%. Phylogenetic analysis classified SgGH3.1 into Group Ⅱ of GH3 family, which have been proven to involve with auxins conjugation. Expression profiling revealed that SgGH3.1 responded rapidly to cold stress in stylo leaves. Overexpression of SgGH3.1 in Arabidopsis thaliana altered sensitivity to exogenous IAA, up-regulated transcription of AtCBF1-3 genes, activated physiological responses against cold stress, and enhanced chilling and cold tolerances. This is the first report of a GH3 gene in stylos, which not only validated its function in IAA homeostasis and cold responses, but also gave insight into breeding of cold-tolerant stylos.

Transcriptome profiling provides new insights into florets number difference of inflorescence in L.angustifolia

Background Lavender flowers essential oil had been used for a variety of therapeutic and cosmetic purposes, and had been popular for centuries. The previous studies of lavender mainly focused on essential oil composition and extraction methods, ignoring the factors that affected the production of essential oils, such as the floret number. This study aims to get a deep insight into the mechanism of florets number difference. Results 1.Hormone profile showed positive correlation between ABA content and the number of florets, while IAA was negatively correlated. 2. RNA-Seq results showed that 2848 differentially expressed genes (DEG) were screened by comparing different florets samples in one plant. By analyzing dynamic changes of differentially expressed genes, many potentially interesting genes that encoded putative regulators or key components of ABA metabolism and signaling transduction were identified, such as NCED, PYL, PP2C, SnRK2. 3.Common network analysis showed that the key genes of ABA pathway metabolism were negatively related to the DEG of IAA, especial IAA18. 4. Exogenous IAA significantly inhibited the number of lavender florets and affected the expression of ABA pathway metabolism genes, such as NCED, PP2C.Conclusions 1. The different concentrations of ABA lead to florets number difference in the L.angustifolia “JX-2” clusters; 2. ABA may affect the florets number by regulating IAA transport and accumulation.The results will be useful for a better understanding of the molecular mechanism on florets number difference, which will lay the foundation for molecular breeding of muti-flortes varieties.

Auxin Homeostasis and Distribution of the Auxin Efflux Carrier PIN2 Require Vacuolar NHX-Type Cation/H+ Antiporter Activity

The Arabidopsis vacuolar Na+/H+ transporters (NHXs) are important regulators of intracellular pH, Na+ and K+ homeostasis and necessary for normal plant growth, development, and stress acclimation. Arabidopsis contains four vacuolar NHX isoforms known as AtNHX1 to AtNHX4. The quadruple knockout nhx1nhx2nhx3nhx4, lacking any vacuolar NHX-type antiporter activity, displayed auxin-related phenotypes including loss of apical dominance, reduced root growth, impaired gravitropism and less sensitivity to exogenous IAA and NAA, but not to 2,4-D. In nhx1nhx2nhx3nhx4, the abundance of the auxin efflux carrier PIN2, but not PIN1, was drastically reduced at the plasma membrane and was concomitant with an increase in PIN2 labeled intracellular vesicles. Intracellular trafficking to the vacuole was also delayed in the mutant. Measurements of free IAA content and imaging of the auxin sensor DII-Venus, suggest that auxin accumulates in root tips of nhx1nhx2nhx3nhx4. Collectively, our results indicate that vacuolar NHX dependent cation/H+ antiport activity is needed for proper auxin homeostasis, likely by affecting intracellular trafficking and distribution of the PIN2 efflux carrier.

Comparative transcriptome analysis revealed the cooperative regulation of sucrose and IAA on adventitious root formation in lotus (Nelumbo nucifera Gaertn)

Background In China, lotus is an important cultivated crop with multiple applications in ornaments, food, and environmental purification. Adventitious roots (ARs), a secondary root is necessary for the uptake of nutrition and water as the lotus principle root is underdeveloped. Therefore, AR formation in seedlings is very important for lotus breeding due to its effect on plant early growth. As lotus ARs formation was significantly affected by sucrose treatment, we analyzed the expression of genes and miRNAs upon treatment with differential concentrations of sucrose, and a crosstalk between sucrose and IAA was also identified. Results Notably, 20 mg/L sucrose promoted the ARs development, whereas 60 mg/L sucrose inhibited the formation of ARs. To investigate the regulatory pathway during ARs formation, the expression of genes and miRNAs was evaluated by high-throughput tag-sequencing. We observed that the expression of 5438, 5184, and 5345 genes was enhanced in the GL20/CK0, GL60/CK0, and CK1/CK0 libraries, respectively. Further, the expression of 73, 78, and 71 miRNAs was upregulated in the ZT20/MCK0, ZT60/MCK0, and MCK1/MCK0 libraries, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the differentially expressed genes and miRNAs in the GL20/GL60 and ZT20/ZT60 libraries were involved in signal transduction. A large number of these genes (29) and miRNAs (53) were associated with plant hormone metabolism. We observed an association between five miRNAs (miR160, miR156a-5p, miR397-5p_1, miR396a and miR167d) and nine genes (auxin response factor, protein brassinosteroid insensitive 1, laccase, and peroxidase 27) in the ZT20/ ZT60 libraries during ARs formation. Quantitative polymerase chain reaction (qRT-PCR) was used to validate the high-throughput tag-sequencing data. Conclusions We found that the expression of many critical genes involved in IAA synthesis and IAA transport was changed after treatment with various concentration of sucrose. Based on the change of these genes expression, IAA and sucrose content, we concluded that sucrose and IAA cooperatively regulated ARs formation. Sucrose affected ARs formation by improving IAA content at induction stage, and increased sucrose content might be also required for ARs development according to the changes tendency after application of exogenous IAA.

TDIF regulates auxin accumulation and modulates auxin sensitivity to enhance both adventitious root and lateral root formation in poplar trees

Of six TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF)-encoding genes in poplar, PtTDIF1 is predominantly expressed in adventitious roots (ARs), and the other five PtTDIFs are preferentially expressed in lateral roots (LRs). Upon auxin application, expression of all PtTDIFs declined in ARs but transiently increased in LRs. Both exogenous TDIF peptides and overexpression of PtTDIFs in poplar positively regulated the initiation and elongation of LRs, and overexpression of PtTDIFs also increased the number of ARs. As visualized by the auxin-responsive marker DR5:GUS, TDIF had differential impacts on the auxin signaling activity in ARs and LRs, which was corroborated by the free indole-3-acetic acid (IAA) measurements in them. Shoot tips of PtTDIF2- and PtTDIFL2-overexpressing (together as PtTDIFsOE) trees revealed an enhanced IAA biosynthetic capacity, and removal of the aerial tissues dramatically diminished the root phenotypes of micro-propagated PtTDIFsOE trees. Furthermore, PtTDIFsOE poplars displayed an increased sensitivity for exogenous IAA, and N-1-naphthylphthalamic acid (NPA) completely blocked the TDIF-induced AR and LR formation. In PtTDIFsOE roots, several auxin-related LR initiation markers such as GATA23, LBD16 and LBD29 were transcriptionally upregulated, further supporting that TDIF regulates LR organogenesis by strengthening the spatiotemporal auxin cues and that dynamic interplays between hormones govern root branching and developmental plasticity in tree species.

Indole -3-acetic acid improves drought tolerance of white clover via activating auxin, abscisic acid and jasmonic acid related genes and inhibiting senescence genes

Background: Auxin may have a positive effect on plants under drought stress. White clover is widely cultivated and often prone to water shortages. In the present study, we investigated the effects of exogenous indole -3-acetic acid (IAA) on growth and physiological changes of white clover under drought stress condition. The contents of endogenous IAA and other hormones including ABA, CTK, JA, GA, IAA, and SA were assayed. Moreover, expressions of auxin-responsive genes, drought-responsive genes and leaf senescence-associated genes were detected in response to exogenous IAA. Results: Compared to control, drought stress alone significantly diminished stem dry weigh, relative water content (RWC) and total chlorophyll content (Chl). Exogenous IAA treatment significantly increased RWC and Chl, whereas L-AOPP treatment drastically decreased stem dry weight, RWC and Chl under drought stress condition. Additionally, exogenous IAA treatment significantly increased ABA content and JA content, up-regulated expression of auxin responsive genes ( GH3.1 , GH3.9 , IAA8 ), drought stress responsive genes ( bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 and RD22 ), and down-regulated expressions of auxin-responding genes ( GH3.3 , GH3.6 , IAA27 ) and leaf senescence genes ( SAG101 and SAG102 ) in the presence of PEG. Contrarily, L-AOPP treatment significantly reduced contents of ABA, GA3 and JA, down-regulated expressions of GH3.1 , GH3.9 , IAA8 , bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 , ERD and RD22 , and up-regulated SAG101 and SAG102 . Conclusions: Exogenous IAA improved drought tolerance of white clover possibly due to endogenous plant hormone concentration changes and modulation of genes involving in drought stress response and leaf senescence. These results provided useful information to understand mechanisms of IAA improved drought tolerance in white clover.

Indole -3-acetic acid improves drought tolerance of white clover associated with activating auxin-related genes, abscisic acid and jasmonic acid-induced stress responsive transcription factors, and inhibiting senescence genes

Auxin plays an important role in regulating plant development. To idenfity what effects IAA has in white clover under drought stress and how IAA regulates white clover response to drought. Relative water content, chlorophyll content, six phytohormones content, expression of gene-related to IAA signal perception and synthesis, transcription factor genes related to drought-resistance and senescence genes were investigated in white clover pretreated with 1µM exogenous IAA or 100µM L-AOPP (inhibitor of IAA synthesis) under 15% polyethylene glycol-6000 (PEG-6000). Compared to control, drought stress significantly diminished stem dry weight (g/10 plant), relative water content (RWC, %) and total chlorophyll content (Chl, mg/g). Exogenous IAA significantly increased RWC and Chl, however, L-AOPP decreased stem dry weight, RWC and Chl compared with direct drought treatment. Besides that, compared with direct drought set, exogenous IAA significantly improved ABA content and JA content, up-regulated expression levels of some auxin-responsive genes ( GH3.1 , GH3.9 , IAA8 ) , transcription factor genes ( bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 ) related to drought-resistance and drought resistance gene ( RD22 ). Meanwhile, exogenous IAA down-regulated expression levels of several auxin-responsive genes ( GH3.3 , GH3.6 , IAA27 ) and leaf senescence genes ( SAG101 and SAG102 ). Contrarily, L-AOPP significantly reduced ABA, GA3 and JA content, down-regulated expression levels of GH3.1 , GH3.9 , IAA8 , bZIP11 , DREB2 , MYB14 , MYB48 , WRKY2 , WRKY56 , WRKY108715 , ERD and RD22 , and up-regulated expression levels of SAG101 and SAG102 . Collectively, these data suggested a positive role of exogenous IAA in alleviating drought damage through many involved physiological processes in white clover.