Sorghum, a C4 model plant, has been studied to develop an understanding of the molecular mechanism of resistance to stress. The auxin-response genes, auxin/indole-3-acetic acid (Aux/IAA), auxin-response factor (ARF), Gretchen Hagen3 (GH3), small auxin-up RNAs, and lateral organ boundaries (LBD), are involved in growth/development and stress/defense responses in Arabidopsis and rice, but they have not been studied in sorghum. In the present paper, the chromosome distribution, gene duplication, promoters, intron/exon, and phylogenic relationships of Aux/IAA, ARF, GH3, and LBD genes in sorghum are presented. Furthermore, real-time PCR analysis demonstrated these genes are differently expressed in leaf/root of sorghum and indicated the expression profile of these gene families under IAA, brassinosteroid (BR), salt, and drought treatments. The SbGH3 and SbLBD genes, expressed in low level under natural condition, were highly induced by salt and drought stress consistent with their products being involved in both abiotic stresses. Three genes, SbIAA1, SbGH3-13, and SbLBD32, were highly induced under all the four treatments, IAA, BR, salt, and drought. The analysis provided new evidence for role of auxin in stress response, implied there are cross talk between auxin, BR and abiotic stress signaling pathways.

ABSTRACT

Summary• Auxin has an important role in maintaining optimal root system architecture (RSA) that can cope with growth reductions of crops caused by water or nutrient shortages. However, the mechanism of controlling RSA remains largely unclear. Here, we found a limiting factor of RSA -OsARF12 -an auxin response factor whose knockout led to decreased primary root length in rice (Oryza sativa).• OsARF12 as a transcription activator can facilitate the expression of the auxin response element DR5::GFP, and OsARF12 was inhibited by osa-miRNA167d by transient expression in tobacco and rice callus.• The root elongation zones of osarf12 and osarf12 ⁄ 25, which had lower auxin concentrations, were distinctly shorter than for the wild-type, possibly as a result of decreased expression of auxin synthesis genes OsYUCCAs and auxin efflux carriers OsPINs and OsPGPs. The knockout of OsARF12 also altered the abundance of mitochondrial iron-regulated (OsMIR), iron (Fe)-regulated transporter1 (OsIRT1) and short postembryonic root1 (OsSPR1) in roots of rice, and resulted in lower Fe content.• The data provide evidence for the biological function of OsARF12, which is implicated in regulating root elongation. Our investigation contributes a novel insight for uncovering regulation of RSA and the relationship between auxin response and Fe acquisition.

Auxin response factors (ARFs) are key regulators of plant growth and development. Through interaction with auxin/indole acetic acid (Aux/IAA) proteins, they influence the expression of auxin response genes. An ARF gene family has been predicted in rice, but the functions of the individual structural domains of the OsARFs remain obscure. Bioinformatics was used to analyse the position of the DNA-binding domain (DBD), middle region (MR), and C-terminal dimerization domain (CTD) of OsARFs, and experimentally confirmed the presence of a classical monopartite nuclear localization signal (NLS) in the DBD. The DBD was shown to contribute to nuclear localization of OsARF proteins in addition to its known DNA-binding function. Interactions between 14 integrated OsARFs and 15 OsIAA proteins were tested using yeast two-hybrid assays. It was found that eight OsARF activators interacted with the 15 OsIAA proteins, while six OsARF repressors did not. The interactions between the MR+CTD or CTD of 10 OsARFs and 15 OsIAA proteins were also tested and the results were consistent with those of each intact OsARF, although some slight differences in interaction intensity were observed by α-galactosidase quantitative assays. The truncated CTD of OsARF11 did not interact with any OsIAA, implying that the CTD is required for ARF–IAA dimerization, and that the MR influences the interaction intensity in yeast. A subset of the interactions in yeast were also observed in tobacco plants using firefly luciferase complementation imaging assays, indicating that these interactions are specific in plants, and might have a special role in the auxin signalling response. This study provides new insight into the structure of OsARF proteins and ARF–Aux/IAA interactions.

Auxin is transported by the influx carriers auxin resistant 1/like aux1 (AUX/LAX), and the efflux carriers pin‐formed (PIN) and P‐glycoprotein (PGP), which play a major role in polar auxin transport. Several auxin transporter genes have been characterized in dicotyledonous Arabidopsis, but most are unknown in monocotyledons, especially in sorghum. Here, we analyze the chromosome distribution, gene duplication and intron/exon of SbPIN, SbLAX and SbPGP gene families, and examine their phylogenic relationships in Arabidopsis, rice and sorghum. Real‐time PCR analysis demonstrated that most of these genes were differently expressed in the organs of sorghum. SbPIN3 and SbPIN9 were highly expressed in flowers, SbLAX2 and SbPGP17 were mainly expressed in stems, and SbPGP7 was strongly expressed in roots. This suggests that individual genes might participate in specific organ development. The expression profiles of these gene families were analyzed after treatment with: (a) the phytohormones indole‐3‐acetic acid and brassinosteroid; (b) the polar auxin transport inhibitors 1‐naphthoxyacetic acids, 1‐naphthylphthalamic acid and 2,3,5‐triiodobenzoic acid; and (c) abscissic acid and the abiotic stresses of high salinity and drought. Most of the auxin transporter genes were strongly induced by indole‐3‐acetic acid and brassinosteroid, providing new evidence for the synergism of these phytohormones. Interestingly, most genes showed similar trends in expression under polar auxin transport inhibitors and each also responded to abscissic acid, salt and drought. This study provides new insights into the auxin transporters of sorghum.

Plant responses to auxin and phosphate (Pi) starvation are closely linked. However, the underlying mechanisms connecting auxin to phosphate starvation (-Pi) responses are largely unclear. Here, we show that OsARF16, an auxin response factor, functions in both auxin and -Pi responses in rice (Oryza sativa L.). The knockout of OsARF16 led to primary roots (PR), lateral roots (LR) and root hair losing sensitivity to auxin and -Pi response. OsARF16 expression and OsARF16::GUS staining in PR and LR of rice Nipponbare (NIP) were induced by indole acetic acid and -Pi treatments. In -Pi conditions, the shoot biomass of osarf16 was slightly reduced, and neither root growth nor iron content was induced, indicating that the knockout of OsARF16 led to loss of response to Pi deficiency in rice. Six phosphate starvation-induced genes (PSIs) were less induced by -Pi in osarf16 and these trends were similar to a knockdown mutant of OsPHR2 or AtPHR1, which was a key regulator under -Pi. These data first reveal the biological function of OsARF16, provide novel evidence of a linkage between auxin and -Pi responses and facilitate the development of new strategies for the efficient utilization of Pi in rice.

SummaryPhosphorus (P) is crucial nutrient element for crop growth and development. However, the network pathway regulating homeostasis of phosphate (Pi) in crops has many molecular breeding unknowns. Here, we report that an auxin response factor, OsARF12, functions in Pi homeostasis.Measurement of element content, quantitative reverse transcription polymerase chain reaction analysis and acid phosphatases (APases) activity assay showed that the osarf12 mutant and osarf12/25 double mutant with P-intoxicated phenotypes had higher P concentrations, up-regulation of the Pi transporter encoding genes and increased APase activity under Pi-sufficient/-deficient (+Pi/ÀPi, 0.32/0 mM NaH 2 PO 4 ) conditions.Transcript analysis revealed that Pi-responsive genes -Phosphate starvation (OsIPS)1 and OsIPS2, SYG1/Pho81/XPR1(OsSPX1), Sulfoquinovosyldiacylglycerol 2 (OsSQD2), R2R3 MYB transcription factor (OsMYB2P-1) and Transport Inhibitor Response1 (OsTIR1) -were more abundant in the osarf12 and osarf12/25 mutants under +Pi/ÀPi conditions. Knockout of OsARF12 also influenced the transcript abundances of the OsPHR2 gene and its downstream components, such as OsMiR399j, OsPHO2, OsMiR827, OsSPX-MFS1 and OsSPX-MFS2. Results from -Pi/1-naphthylphthalamic acid (NPA) treatments, and auxin reporter DR5::GUS staining suggest that root system alteration and Pi-induced auxin response were at least partially controlled by OsARF12.These findings enrich our understanding of the biological functions of OsARF12, which also acts in regulating Pi homeostasis.

These authors contributed equally to this work. SUMMARYMembers of the ATP Binding Cassette B/Multidrug-Resistance/P-glyco-protein (ABCB/MDR/PGP) subfamily were shown to function primarily in Oryza sativa (rice) auxin transport; however, none of the rice ABCB transporters have been functionally characterized. Here, we describe that a knock-down of OsABCB14 confers decreased auxin concentrations and polar auxin transport rates, conferring insensitivity to 2,4-dichlorophenoxyacetic acid (2,4-D) and indole-3-acetic acid (IAA). OsABCB14 displays enhanced specific auxin influx activity in yeast and protoplasts prepared from rice knock-down alleles. OsABCB14 is localized at the plasma membrane, pointing to an important directionality under physiological conditions. osabcb14 mutants were surprisingly found to be insensitive to iron deficiency treatment (-Fe). Their Fe concentration is higher and upregulation of Fe deficiency-responsive genes is lower in osabcb14 mutants than in wild-type rice (Nipponbare, NIP). Taken together, our results strongly support the role of OsABCB14 as an auxin influx transporter involved in Fe homeostasis. The functional characterization of OsABCB14 provides insights in monocot auxin transport and its relationship to Fe nutrition.