Overexpression of MsGH3.5 inhibits shoot and root development through the auxin and cytokinin pathways in apple plants

Phytohormonal interactions are crucial for plant development. Auxin and cytokinin (CK) both play critical roles in regulating plant growth and development; however, the interaction between these two phytohormones is complex and not fully understood. Here, we isolated a wild apple (Malus sieversii Roem) GRETCHEN HAGEN3 (GH3) gene, MsGH3.5, encoding an indole‐3‐acetic acid (IAA)‐amido synthetase. Overexpression of MsGH3.5 significantly reduced the free IAA content and increased the content of some IAA‐amino acid conjugates, and MsGH3.5‐overexpressing lines were dwarfed and produced fewer adventitious roots (ARs) than the control. This phenotype is consistent with the role of GH3 in conjugating excess free active IAA to amino acids in auxin homeostasis. Surprisingly, overexpression of MsGH3.5 significantly increased CK concentrations in the whole plant, and altered the expression of genes involved in CK biosynthesis, metabolism and signaling. Furthermore, exogenous CK application induced MsGH3.5 expression through the activity of the CK type‐B response regulator, MsRR1a, which mediates the CK primary response. MsRR1a activated MsGH3.5 expression by directly binding to its promoter, linking auxin and CK signaling. Plants overexpressing MsRR1a also displayed fewer ARs, in agreement with the regulation of MsGH3.5 expression by MsRR1a. Taken together, we reveal that MsGH3.5 affects apple growth and development by modulating auxin and CK levels and signaling pathways. These findings provide insight into the interaction between the auxin and CK pathways, and might have substantial implications for efforts to improve apple architecture.     

The role of strigolactones in photomorphogenesis of pea is limited to adventitious rooting

The recently discovered group of plant hormones, the strigolactones, have been implicated in regulating photomorphogenesis. We examined this extensively in our strigolactone synthesis and response mutants and could find no evidence to support a major role for strigolactone signaling in classic seedling photomorphogenesis (e.g. elongation and leaf expansion) in pea (Pisum sativum), consistent with two recent independent reports in Arabidopsis. However, we did find a novel effect of strigolactones on adventitious rooting in darkness. Strigolactone‐deficient mutants, Psccd8 and Psccd7, produced significantly fewer adventitious roots than comparable wild‐type seedlings when grown in the dark, but not when grown in the light. This observation in dark‐grown plants did not appear to be due to indirect effects of other factors (e.g. humidity) as the constitutively de‐etiolated mutant, lip1, also displayed reduced rooting in the dark. This role for strigolactones did not involve the MAX2 F‐Box strigolactone response pathway as Psmax2 f‐box mutants did not show a reduction in adventitious rooting in the dark compared with wild‐type plants. The auxin‐deficient mutant bushy also reduced adventitious rooting in the dark, as did decapitation of wild‐type plants. Rooting was restored by the application of indole‐3‐acetic acid (IAA) to decapitated plants, suggesting a role for auxin in the rooting response. However, auxin measurements showed no accumulation of IAA in the epicotyls of wild‐type plants compared with the strigolactone synthesis mutant Psccd8, suggesting that changes in the gross auxin level in the epicotyl are not mediating this response to strigolactone deficiency.     

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11‐controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root‐to‐shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL‐related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low‐K‐enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11‐regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K‐deficiency‐enhanced expression of several RR genes encoding type‐A cytokinin‐responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K‐deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.     

OsRPK1, a novel leucine-rich repeat receptor-like kinase,negatively regulates polar auxin transport and root development in rice

Background

Leucine-rich-repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of putative RLKs in plants. Although several members in this subfamily have been identified, the studies about the relationships between LRR-RLKs and root development are still few. We previously identified a novel LRR-RLK in rice roots, and named it OsRPK1.

Methods

In this study, we first detected OsRPK1 kinase activity in vitro, and assessed its expression profile. We then investigated its biological function using transgenic rice plants over- and under-expressing OsRPK1.

Results

The OsRPK1 gene, which encodes a Ca^2 +-independent Ser/Thr kinase, was predominantly expressed in root tips, leaf blades, and undifferentiated suspension cells, and was markedly induced by treatment with auxin or ABA. Knockdown of OsRPK1 promoted the growth of transgenic rice plants, and increased plant height and tiller numbers. In contrast, over-expressing plants showed undeveloped adventitious roots, lateral roots, and a reduced root apical meristem. OsRPK1 over-expression also inhibited the expression of most auxin efflux carrier OsPIN genes, which was accompanied by changes in PAT and endogenous free IAA distribution in the leaves and roots.

Conclusions

The data indicated that OsRPK1, a novel leucine-rich-repeat receptor-like kinase, affects the root system architecture by negatively regulating polar auxin transport in rice.

General significance

This study demonstrated a common regulatory pathway of root system development in higher plants, which might be initiated by external stimuli via upstream receptor-like kinases and downstream carriers for polar auxin transport.     

Expression and regulation of theRSI-1 gene during lateral root initiation

The tomato geneRSI-1 was previously identified as a molecular marker for auxin-induced lateral root initiation. We have further characterized the expression mode of theRSI-1 gene in tomato andArabidopsis thaliana. Northern blot analyses revealed that the gene was induced specifically by auxin in tomato roots and hypocotyls. For experiments with transgenic plants, the 5′ flanking region of theRSI-1 gene was linked to a GUS reporter gene, then transformed into tomato andArabidopsis. In these transgenic tomato plants, GUS activity was detected at the sites of initiation for lateral and adventitious roots. Expression of the fusion gene was auxin-dependent and tissue-specific. This was consistent with results from the northern blot analyses. In transgenicArabidopsis, the overall expression pattern of theRSI-GUS gene, including tissue specificity and auxin inducibility, was comparable to that in transgenic tomato seedlings. These results indicate that an identical regulatory mechanism for lateral root initiation might be conserved in both plants. Thus, the expression mode of theRSI-CUS gene inArabidopsis mutants defective in lateral root development should be investigated to provide details of this process.     

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root‐predominant gene promoter (SbUGT) to drive the expression of a tryptophan‐2‐monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild‐type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild‐type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.     

Transgenic white clover. Studies with the auxin-responsive promoter,GH3, in root gravitropism and lateral root development

We report an improved method for white clover (Trifolium repens) transformation usingAgrobacterium tumefaciens. High efficiencies of transgenic plant production were achieved using cotyledons of imbibed mature seed. Transgenic plants were recovered routinely from over 50% of treated cotyledons. Thebar gene and phosphinothricin selection was shown to be a more effective selection system thannptII (kanamycin selection) oraadA (spectinomycin selection). White clover was transformed with the soybean auxin responsive promoter, GH3, fused to the GUS gene (-glucuronidase) to study the involvement of auxin in root development. Analysis of 12 independent transgenic plants showed that the location and pattern of GUS expression was consistent but the levels of expression varied. The level of GH3:GUS expression in untreated plants was enhanced specifically by auxin-treatment but the pattern of expression was not altered. Expression of the GH3:GUS fusion was not enhanced by other phytohormones. A consistent GUS expression pattern was evident in untreated plants presumably in response to endogenous auxin or to differences in auxin sensitivity in various clover tissues. In untreated plants, the pattern of GH3:GUS expression was consistent with physiological responses which are regarded as being auxin-mediated. For the first time it is shown that localised spots of GH3:GUS activity occurred in root cortical tissue opposite the sites where lateral roots subsequently were initiated. Newly formed lateral roots grew towards and through these islands of GH3:GUS expression, implying the importance of auxin in controlling lateral root development. Similarly, it is demonstrated for the first time that gravistimulated roots developed a rapid (within 1 h) induction of GH3:GUS activity in tissues on the non-elongating side of the responding root and this induction occurred concurrently with root curvature. These transgenic plants could be useful tools in determining the physiological and biochemical changes that occur during auxin-mediated responses.     

Regulatory role of auxin in adventitious root formation in two species of Rumex,differing in their sensitivity to waterlogging

Adventitious rooting in Rumex plants, in which the root systems were in hypoxic conditions, differed considerably between two species. R. palustris, a species from frequently flooded river forelands, developed a large number of adventitious roots during hypoxia, whereas adventitious root formation was poor in R. thyrsiflorus, a species from seldom flooded dykes and river dunes. Adventitious rooting could also be evoked in aerated plants of both species by application of auxin (1-naphthaleneacetic acid or indoleacetic acid) to the leaves. The response to auxin was dose-dependent, but even high auxin doses could not stimulate R. thyrsiflorus to produce as many adventitious roots as R. palustris. Consequently, the difference between the species in the amount of adventitious root formation was probably genetically determined, and not a result of a different response to auxin. A prerequisite for hypoxia-induced adventitious root formation is the basipetal transport of auxin within the shoot, as specific inhibition of this transport by N-1-naphthylphthalamic acid severely decreased the number of roots in hypoxia-treated plants. It is suggested that hypoxia of the root system causes stagnation of auxin transport in the root system. This can lead to an accumulation of auxin at the base of the shoot rosette, resulting in adventitious root formation.     

The peu‐miR160a−PeARF17.1/PeARF17.2 module participates in the adventitious root development of poplar

Deep roots give rise to flourishing leaves, and the two complement each other. However, the genetic mechanisms underlying adventitious rooting for forest trees have remained elusive. In this study, we verified that peu‐miR160a targets six poplar genes AUXIN RESPONSE FACTORS (ARFs), PeARF10.1, PeARF16.1, PeARF16.2, PeARF16.3, PeARF17.1 and PeARF17.2, using 5’RLM‐RACE. Interaction experiments with peu‐miR160a and PeARFs in poplar protoplasts further confirmed that peu‐miR160a targets and negatively regulates the six PeARFs. Peu‐miR160a and its target genes exhibited obvious temporal expression in different stages of adventitious root development, and they could also be induced by IAA and abscisic acid. Peu‐miR160a‐overexpressing lines exhibited a significant shortening of adventitious root length, an increase in the number of lateral roots, severe dwarfing and shortened internodes. In addition, the overexpression of PeARF17.1 or mPeARF17.2 (peu‐miR160a‐resistant version of PeARF17.2) significantly increased the number of adventitious roots. Furthermore, PeARF17.1‐overexpressing lines had multiple branches with no visible trunk, although the adventitious root length of the PeARF17.1‐overexpressing lines was significantly increased. Our findings reveal that the peu‐miR160a ? PeARF17.1/PeARF17.2 module is an important regulator involved in the development of the adventitious roots of poplar.     

Production of transgenic Aralia elata regenerated from Agrobacterium rhizogenes-mediated transformed roots

Transgenic hairy roots were induced from petiole and root segments of in vitro plant Aralia elata, a medicinal woody shrub, after co-cultivation with A. rhizogenes ATCC 15834. The percentage of putative hairy root induction from root segments was higher (26.7%) than petiole explants (10.0%). Hairy roots showed active production of lateral roots with vigorous elongation. Transgenic plants were regenerated from hairy roots via somatic embryogenesis. These plants had wrinkled leaves, short petioles and numerous lateral hairy roots. The RT-PCR analysis showed the expression of rol A, B, C, D, aux 1 and 2 genes differed between the transgenic lines. Endogenous IAA level was higher in transgenic than non-transgenic plants. Conclusively, transgenic hairy roots were developed for first time in A. elata and the transgenic hairy root lines showed distinct morphological growth pattern and gene expression.     

OsCYP2, a chaperone involved in degradation of auxin‐responsive proteins,plays crucial roles in rice lateral root initiation

Auxin plays a pivotal role in many facets of plant development. It acts by inducing the interaction between auxin‐responsive [auxin (AUX)/indole‐3‐acetic acid (IAA)] proteins and the ubiquitin protein ligase SCF^TIR to promote the degradation of the AUX/IAA proteins. Other cofactors and chaperones that participate in auxin signaling remain to be identified. Here, we characterized rice (Oryza sativa) plants with mutations in a cyclophilin gene (OsCYP2). cyp2 mutants showed defects in auxin responses and exhibited a variety of auxin‐related growth defects in the root. In cyp2 mutants, lateral root initiation was blocked after nuclear migration but before the first anticlinal division of the pericycle cell. Yeast two‐hybrid and in vitro pull‐down results revealed an association between OsCYP2 and the co‐chaperone Suppressor of G2 allele of skp1 (OsSGT1). Luciferase complementation imaging assays further supported this interaction. Similar to previous findings in an Arabidopsis thaliana SGT1 mutant (atsgt1b), degradation of AUX/IAA proteins was retarded in cyp2 mutants treated with exogenous 1‐naphthylacetic acid. Our results suggest that OsCYP2 participates in auxin signal transduction by interacting with OsSGT1.     

Influence of galactoglucomannan oligosaccharides on root culture of <Emphasis Type="Italic">Karwinskia humboldtiana</Emphasis>

Galactoglucomannan oligosaccharides (GGMOs) activity in K. humboldtiana root culture has been determined. GGMOs inhibited adventitious root growth and lateral root induction in contrast to IAA, IBA, and NAA stimulating effect in these processes. Similarly, the combination of GGMOs with natural auxins (IAA, IBA) evoked an inhibition of adventitious root growth and lateral root induction that depended on the oligosaccharides concentration and the type of auxin. The growth stimulating effect of the synthetic auxin, NAA, in adventitious roots was negatively affected by GGMOs, but they were without influence on lateral root induction. The presence of oligosaccharides triggered lateral root position on adventitious roots and the anatomy of adventitious roots (diameter, proportion of primary cortex to the central cylinder, number and size of primary cortical cells, intercellular spaces, and the number of starch grains in cells of primary cortex) in dependence on their coactions with auxin.     

Gibberellins inhibit adventitious rooting in hybrid aspen and Arabidopsis by affecting auxin transport

Knowledge of processes involved in adventitious rooting is important to improve both fundamental understanding of plant physiology and the propagation of numerous plants. Hybrid aspen (Populus tremula × tremuloïdes) plants overexpressing a key gibberellin (GA) biosynthesis gene (AtGA20ox1) grow rapidly but have poor rooting efficiency, which restricts their clonal propagation. Therefore, we investigated the molecular basis of adventitious rooting in Populus and the model plant Arabidopsis. The production of adventitious roots (ARs) in tree cuttings is initiated from the basal stem region, and involves the interplay of several endogenous and exogenous factors. The roles of several hormones in this process have been characterized, but the effects of GAs have not been fully investigated. Here, we show that a GA treatment negatively affects the numbers of ARs produced by wild‐type hybrid aspen cuttings. Furthermore, both hybrid aspen plants and intact Arabidopsis seedlings overexpressing AtGA20ox1, PttGID1.1 or PttGID1.3 genes (with a 35S promoter) produce few ARs, although ARs develop from the basal stem region of hybrid aspen and the hypocotyl of Arabidopsis. In Arabidopsis, auxin and strigolactones are known to affect AR formation. Our data show that the inhibitory effect of GA treatment on adventitious rooting is not mediated by perturbation of the auxin signalling pathway, or of the strigolactone biosynthetic and signalling pathways. Instead, GAs appear to act by perturbing polar auxin transport, in particular auxin efflux in hybrid aspen, and both efflux and influx in Arabidopsis.