Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana.

Aerobic reactions are essential to sustain plant growth and development. Impaired oxygen availability due to excessive water availability, e.g., during waterlogging or flooding, reduces plant productivity and survival. Consequently, plants monitor oxygen availability to adjust growth and metabolism accordingly. Despite the identification of central components in hypoxia adaptation in recent years, molecular pathways involved in the very early activation of low-oxygen responses are insufficiently understood. Here, we characterized three endoplasmic reticulum (ER)-anchored Arabidopsis ANAC transcription factors, namely ANAC013, ANAC016, and ANAC017, which bind to the promoters of a subset of hypoxia core genes (HCGs) and activate their expression. However, only ANAC013 translocates to the nucleus at the onset of hypoxia, i.e., after 1.5 h of stress. Upon hypoxia, nuclear ANAC013 associates with the promoters of multiple HCGs. Mechanistically, we identified residues in the transmembrane domain of ANAC013 to be essential for transcription factor release from the ER, and provide evidence that RHOMBOID-LIKE 2 (RBL2) protease mediates ANAC013 release under hypoxia. Release of ANAC013 by RBL2 also occurs upon mitochondrial dysfunction. Consistently, like ANAC013 knockdown lines, rbl knockout mutants exhibit impaired low-oxygen tolerance. Taken together, we uncovered an ER-localized ANAC013-RBL2 module, which is active during the initial phase of hypoxia to enable fast transcriptional reprogramming.

Hypoxia reshapes Arabidopsis root architecture by integrating ERF-VII factor response and abscisic acid homoeostasis.

Oxygen limitation (hypoxia), arising as a key stress factor due to flooding, negatively affects plant development. Consequently, maintaining root growth under such stress is crucial for plant survival, yet we know little about the root system's adaptions to low-oxygen conditions and its regulation by phytohormones. In this study, we examine the impact of hypoxia and, herein, the regulatory role of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors on root growth in Arabidopsis. We found lateral root (LR) elongation to be actively maintained by hypoxia via ERFVII factors, as erfVII seedlings possess hypersensitivity towards hypoxia regarding their LR growth. Pharmacological inhibition of abscisic acid (ABA) biosynthesis revealed ERFVII-driven counteraction of hypoxia-induced inhibition of LR formation in an ABA-dependent manner. However, postemergence LR growth under hypoxia mediated by ERFVIIs was independent of ABA. In roots, ERFVIIs mediate, among others, the induction of ABA-degrading ABA 8'-hydroxylases CYP707A1 expression. RAP2.12 could activate the pCYC707A1:LUC reporter gene, indicating, combined with single mutant analyses, that this transcription factor regulates ABA levels through corresponding transcript upregulation. Collectively, hypoxia-induced adaptation of the Arabidopsis root system is shaped by developmental reprogramming, whereby ERFVII-dependent promotion of LR emergence, but not elongation, is partly executed through regulation of ABA degradation.

A quantitative trait locus conferring flood tolerance to deepwater rice regulates the formation of two distinct types of aquatic adventitious roots.

A key trait conferring flood tolerance is the ability to grow adventitious roots as a response to submergence. The genetic traits of deepwater rice determining the development and characteristics of aquatic adventitious roots (AAR) had not been evaluated. We used near-isogenic lines introgressed to test the hypothesis that the impressive shoot elongation ability of deepwater rice linked to quantitative trait loci 1 and 12 also promote the development of AAR. The deepwater rice genotype NIL-12 possessed expanded regions at the stem nodes where numerous AAR developed as a response to submergence. Two types (AR1 and AR2) of roots with distinct timing of emergence and large differences in morphological and anatomical traits formed within 3 (AR1) to 7 (AR2) d of submergence. The mechanical impedance provided by the leaf sheath caused AR2 to emerge later promoting thicker roots, higher elongation capacity and higher desiccation tolerance. Upregulation of key genes suggests a joint contribution in activating the meristem in AAR enhancing the development of these in response to submergence. The morphological and anatomical traits suggested that AR2 is better adapted to long-term flooding than AR1. We therefore propose that AR2 in deepwater rice functions as an evolutionary defence strategy to tackle periodic submergence.

Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

Regulation of root adaptive anatomical and morphological traits during low soil oxygen.

Flooding causes oxygen deprivation in soils. Plants adapt to low soil oxygen availability by changes in root morphology, anatomy, and architecture to maintain root system functioning. Essential traits include aerenchyma formation, a barrier to radial oxygen loss, and outgrowth of adventitious roots into the soil or the floodwater. We highlight recent findings of mechanisms of constitutive aerenchyma formation and of changes in root architecture. Moreover, we use modelling of internal aeration to demonstrate the beneficial effect of increasing cortex-to-stele ratio on sustaining root growth in waterlogged soils. We know the genes for some of the beneficial traits, and the next step is to manipulate these genes in breeding in order to enhance the flood tolerance of our crops.

Phytosulfokine (PSK) precursor processing by subtilase SBT3.8 and PSK signaling improve drought stress tolerance in Arabidopsis.

Increasing drought stress poses a severe threat to agricultural productivity. Plants, however, have evolved numerous mechanisms to cope with such environmental stress. Here we report that the stress-induced production of a peptide signal contributes to stress tolerance. The expression of phytosulfokine (PSK) peptide precursor genes, and transcripts of three subtilisin-like serine proteases, SBT1.4, SBT3.7, and SBT3.8, were found to be up-regulated in response to osmotic stress. Stress symptoms were more pronounced in sbt3.8 loss-of-function mutants and could be alleviated by PSK treatment. Osmotic stress tolerance was improved in plants overexpressing the PSK1 precursor (proPSK1) or SBT3.8, resulting in higher fresh weight and improved lateral root development in transgenic plants compared with wild-type plants. We further showed that SBT3.8 is involved in the biogenesis of the bioactive PSK peptide. ProPSK1 was cleaved by SBT3.8 at the C-terminus of the PSK pentapeptide. Processing by SBT3.8 depended on the aspartic acid residue directly following the cleavage site. ProPSK1 processing was impaired in the sbt3.8 mutant. The data suggest that increased expression of proPSK1 in response to osmotic stress followed by the post-translational processing of proPSK1 by SBT3.8 leads to the production of PSK as a peptide signal for stress mitigation.

Tyrosylprotein sulfotransferase-dependent and -independent regulation of root development and signaling by PSK LRR receptor kinases in Arabidopsis.

Tyrosine-sulfated peptides are key regulators of plant growth and development. The disulfated pentapeptide phytosulfokine (PSK) mediates growth via leucine-rich repeat receptor-like kinases, PSKR1 and PSKR2. PSK receptors (PSKRs) are part of a response module at the plasma membrane that mediates short-term growth responses, but downstream signaling of transcriptional regulation remains unexplored. In Arabidopsis, tyrosine sulfation is catalyzed by a single-copy gene (TPST; encoding tyrosylprotein sulfotransferase). We performed a microarray-based transcriptome analysis in the tpst-1 mutant background that lacks sulfated peptides to identify PSK-regulated genes and genes that are regulated by other sulfated peptides. Of the 169 PSK-regulated genes, several had functions in root growth and development, in agreement with shorter roots and a higher lateral root density in tpst-1. Further, tpst-1 roots developed higher numbers of root hairs, and PSK induced expression of WEREWOLF (WER), its paralog MYB DOMAIN PROTEIN 23 (MYB23), and At1g66800 that maintain non-hair cell fate. The tpst-1 pskr1-3 pskr2-1 mutant showed even shorter roots, and higher lateral root and root hair density than tpst-1, revealing unexpected synergistic effects of ligand and PSKR deficiencies. While residual activities may exist, overexpression of PSKR1 in the tpst-1 background induced root growth, suggesting that PSKR1 may be active in the absence of sulfated ligands.

Oxygen in the air and oxygen dissolved in the floodwater both sustain growth of aquatic adventitious roots in rice.

Flooding is an environmental stress that leads to a shortage of O2 that can be detrimental for plants. When flooded, deepwater rice grow floating adventitious roots to replace the dysfunctional soil-borne root system, but the features that ensure O2 supply and hence growth of aquatic roots have not been explored. We investigate the sources of O2 in aquatic adventitious roots and relate aerenchyma and barriers for gas diffusion to local O2 gradients, as measured by microsensor technology, to link O2 distribution in distinct root zones to their anatomical features. The mature root part receives O2 exclusively from the stem. It has aerenchyma that, together with suberin and lignin depositions at the water-root and cortex-stele interfaces, provides a path for longitudinal O2 movement toward the tip. The root tip has no diffusion barriers and receives O2 from the stem and floodwater, resulting in improved aeration of the root tip over mature tissues. Local formation of aerenchyma and diffusion barriers in the mature root channel O2 towards the tip which also obtains O2 from the floodwater. These features explain aeration of floating roots and their ability to grow under water.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Control of Adventitious Root Architecture in Rice by Darkness, Light, and Gravity.

Rice (Oryza sativa) is a semiaquatic plant that is well adapted to partial flooding. Rice stems develop adventitious root (AR) primordia at each node that slowly mature but emerge only when the plant gets flooded, leading to the formation of a whole new secondary root system upon flooding. AR growth is induced by ethylene that accumulates in submerged plant tissues due to its lowered diffusion rate in water. Here, we report that the architecture of the secondary root system in flooded rice plants is controlled not only by altered gas diffusion but also by gravity and light. While ethylene promotes the emergence and growth of ARs, gravity and light determine their gravitropic setpoint angle (i.e. the deviation of growth direction relative to vertical). ARs grow upward at about 120° in the dark and downward at 54° in the light. The upward growth direction is conserved in indica and japonica rice varieties, suggestive of a conserved trait in rice. Experiments with a klinostat and with inverted stem orientation revealed that gravity promotes upward growth by about 10°. Red, far-red, and blue light lead to negative phototropism in a dose-dependent manner, with blue light being most effective, indicating that phytochrome and blue light signaling control AR system architecture. The cpt1 (coleoptile phototropism1) mutant, which lacks one of the phototropin-interacting CPT proteins, shows reduced sensitivity to blue light. Hence, the gravitropic setpoint angle of rice ARs is controlled by genetic and environmental factors that likely balance the need for oxygen supply (upward growth) with avoidance of root desiccation (downward growth).

Sulfated plant peptide hormones.

Sulfated peptides are plant hormones that are active at nanomolar concentrations. The sulfation at one or more tyrosine residues is catalysed by tyrosylprotein sulfotransferase (TPST), which is encoded by a single-copy gene. The sulfate group is provided by the co-substrate 3´-phosphoadenosine 5´-phosphosulfate (PAPS), which links synthesis of sulfated signaling peptides to sulfur metabolism. The precursor proteins share a conserved DY-motif that is implicated in specifying tyrosine sulfation. Several sulfated peptides undergo additional modification such as hydroxylation of proline and glycosylation of hydroxyproline. The modifications render the secreted signaling molecules active and stable. Several sulfated signaling peptides have been shown to be perceived by leucine-rich repeat receptor-like kinases (LRR-RLKs) but have signaling pathways that, for the most part, are yet to be elucidated. Sulfated peptide hormones regulate growth and a wide variety of developmental processes, and intricately modulate immunity to pathogens. While basic research on sulfated peptides has made steady progress, their potential in agricultural and pharmaceutical applications has yet to be explored.

Root Bending Is Antagonistically Affected by Hypoxia and ERF-Mediated Transcription via Auxin Signaling.

When plants encounter soil water logging or flooding, roots are the first organs to be confronted with reduced gas diffusion resulting in limited oxygen supply. Since roots do not generate photosynthetic oxygen, they are rapidly faced with oxygen shortage rendering roots particularly prone to damage. While metabolic adaptations to low oxygen conditions, which ensure basic energy supply, have been well characterized, adaptation of root growth and development have received less attention. In this study, we show that hypoxic conditions cause the primary root to grow sidewise in a low oxygen environment, possibly to escape soil patches with reduced oxygen availability. This growth behavior is reversible in that gravitropic growth resumes when seedlings are returned to normoxic conditions. Hypoxic root bending is inhibited by the group VII ethylene response factor (ERFVII) RAP2.12, as rap2.12-1 seedlings show exaggerated primary root bending. Furthermore, overexpression of the ERFVII member HRE2 inhibits root bending, suggesting that primary root growth direction at hypoxic conditions is antagonistically regulated by hypoxia and hypoxia-activated ERFVIIs. Root bending is preceded by the establishment of an auxin gradient across the root tip as quantified with DII-VENUS and is synergistically enhanced by hypoxia and the auxin transport inhibitor naphthylphthalamic acid. The protein abundance of the auxin efflux carrier PIN2 is reduced at hypoxic conditions, a response that is suppressed by RAP2.12 overexpression, suggesting antagonistic control of auxin flux by hypoxia and ERFVII. Taken together, we show that hypoxia triggers an escape response of the primary root that is controlled by ERFVII activity and mediated by auxin signaling in the root tip.

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1.

Phytosulfokine (PSK) is perceived by the leucine-rich repeat receptor kinase PSKR1 and promotes growth in Arabidopsis thaliana. PSKR1 is coexpressed with the CYCLIC NUCLEOTIDE-GATED CHANNEL gene CNGC17. PSK promotes protoplast expansion in the wild type but not in cngc17. Protoplast expansion is likewise promoted by cGMP in a CNGC17-dependent manner. Furthermore, PSKR1-deficient protoplasts do not expand in response to PSK but are still responsive to cGMP, suggesting that cGMP acts downstream of PSKR1. Mutating the guanylate cyclase center of PSKR1 impairs seedling growth, supporting a role for PSKR1 signaling via cGMP in planta. While PSKR1 does not interact directly with CNGC17, it interacts with the plasma membrane-localized H(+)-ATPases AHA1 and AHA2 and with the BRI-associated receptor kinase 1 (BAK1). CNGC17 likewise interacts with AHA1, AHA2, and BAK1, suggesting that PSKR1, BAK1, CNGC17, and AHA assemble in a functional complex. Roots of deetiolated bak1-3 and bak1-4 seedlings were unresponsive to PSK, and bak1-3 and bak1-4 protoplasts expanded less in response to PSK but were fully responsive to cGMP, indicating that BAK1 acts in the PSK signal pathway upstream of cGMP. We hypothesize that CNGC17 and AHAs form a functional cation-translocating unit that is activated by PSKR1/BAK1 and possibly other BAK1/RLK complexes.

Phosphorylation of the phytosulfokine peptide receptor PSKR1 controls receptor activity.

The phytosulfokine peptide receptor PSKR1 is modified by phosphorylation of its cytoplasmic kinase domain. We analyzed defined phosphorylation sites by site-directed mutagenesis with regard to kinase activity in vitro and receptor activity in planta. S696 and S698 in the juxtamembrane (JM) domain are phosphorylated in planta. The phosphomimetic S696D/S698D replacements resulted in reduced transphosphorylation activity of PSKR1 kinase in vitro but did not reduce autophosphorylation activity. Growth-promoting activity of the PSKR1(S696D/S698D) receptor isoform was impaired in the shoot but not in the root. The JM domain thus seems to be important for phosphorylation of a target protein required for shoot growth promotion. The phosphomimetic replacement T998D at the C-terminus (CT) abolished kinase activity in vitro but not receptor function in planta, indicating that additional levels of regulation exist in planta. A possible mode of receptor regulation is the interaction with regulatory proteins such as the calcium sensor calmodulin (CaM). We show that the previously reported binding of CaM2 to PSKR1 is calcium-dependent, occurs predominately to the hypophosphorylated soluble PSKR1 kinase, and does not significantly change PSKR1 kinase activity. In conclusion, our results show that peptide signaling of growth by PSKR1 is regulated by differential phosphorylation of the juxtamembrane and C-terminal domains of the intracellular receptor part and suggest that interaction of PSKR1 with CaM serves a function other than the regulation of kinase activity.

Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response.

Kinase activity and calmodulin binding are essential for growth signaling by the phytosulfokine receptor PSKR1.

The cell growth-promoting peptide phytosulfokine (PSK) is perceived by leucine-rich repeat (LRR) receptor kinases. To elucidate PSK receptor function we analyzed PSKR1 kinase activity and binding to Ca(2+) sensors and evaluated the contribution of these activities to growth control in planta. Ectopically expressed PSKR1 was capable of auto- and transphosphorylation. Replacement of a conserved lysine within the ATP-binding region by a glutamate resulted in the inhibition of auto- and transphosphorylation kinase activities. Expression of the kinase-inactive PSKR1(K762E) receptor in the pskr null background did not restore root or shoot growth. Instead, the mutant phenotype was enhanced suggesting that the inactive receptor protein exerts growth-inhibitory activity. Bioinformatic analysis predicted a putative calmodulin (CaM)-binding site within PSKR1 kinase subdomain VIa. Bimolecular fluorescence complementation analysis demonstrated that PSKR1 binds to all isoforms of CaM, more weakly to the CaM-like protein CML8 but apparently not to CML9. Mutation of a conserved tryptophan (W831S) within the predicted CaM-binding site strongly reduced CaM binding. Expression of PSKR1(W831S) in the pskr null background resulted in growth inhibition that was similar to that of the kinase-inactive receptor. We conclude that PSK signaling requires Ca(2+) /CaM binding and kinase activity of PSKR1 in planta. We further propose that the inactivated kinase interferes with other growth-promoting signaling pathway(s).

Phytosulfokine peptide signalling.

Phytosulfokine (PSK) belongs to the group of plant peptide growth factors. It is a disulfated pentapeptide encoded by precursor genes that are ubiquitously present in higher plants, suggestive of universal functions. Processing of the preproprotein involves sulfonylation by a tyrosylprotein sulfotransferase in the trans-golgi and proteolytic cleavage in the apoplast. The secreted peptide is perceived at the cell surface by a membrane-bound receptor kinase of the leucine-rich repeat family. The PSK receptor PSKR1 from Arabidopsis thaliana is an active kinase and has guanylate cyclase activity resulting in dual-signal outputs. Receptor activity is regulated by calmodulin. While PSK may be an autocrine growth factor, it also acts non-cell autonomously by promoting growth of cells that are receptor-deficient. In planta, PSK has multiple functions. It promotes cell growth, acts in the quiescent centre cells of the root apical meristem, contributes to funicular pollen tube guidance, and differentially alters immune responses depending on the pathogen. It has been suggested that PSK integrates growth and defence signals to balance the competing metabolic costs of these responses. This review summarizes our current understanding of PSK synthesis, signalling, and activity.

Emerging roots alter epidermal cell fate through mechanical and reactive oxygen species signaling.

A central question in biology is how spatial information is conveyed to locally establish a developmental program. Rice (Oryza sativa) can survive flash floods by the emergence of adventitious roots from the stem. Epidermal cells that overlie adventitious root primordia undergo cell death to facilitate root emergence. Root growth and epidermal cell death are both controlled by ethylene. This study aimed to identify the signal responsible for the spatial control of cell death. Epidermal cell death correlated with the proximity to root primordia in wild-type and ADVENTITIOUS ROOTLESS1 plants, indicating that the root emits a spatial signal. Ethylene-induced root growth generated a mechanical force of ~18 millinewtons within 1 h. Force application to epidermal cells above root primordia caused cell death in a dose-dependent manner and was inhibited by 1-methylcyclopropene or diphenylene iodonium, an inhibitor of NADPH oxidase. Exposure of epidermal cells not overlying a root to either force and ethylene or force and the catalase inhibitor aminotriazole induced ectopic cell death. Genetic downregulation of the reactive oxygen species (ROS) scavenger METALLOTHIONEIN2b likewise promoted force-induced ectopic cell death. Hence, reprogramming of epidermal cell fate by the volatile plant hormone ethylene requires two signals: mechanosensing for spatial resolution and ROS for cell death signaling.

Phytosulfokine peptide signaling controls pollen tube growth and funicular pollen tube guidance in Arabidopsis thaliana.

Phytosulfokine (PSK) is a peptide growth factor that requires tyrosine sulfation carried out by tyrosylprotein sulfotransferase (TPST) for its activity. PSK is processed from precursor proteins encoded by five genes in Arabidopsis thaliana and perceived by receptor kinases encoded by two genes in Arabidopsis. pskr1-3 pskr2-1 and tpst-1 knockout mutants displayed reduced seed production, indicative of a requirement for PSK peptide signaling in sexual plant reproduction. Expression analysis revealed PSK precursor and PSK receptor gene activity in reproductive organs with strong expression of PSK2 in pollen. In support of a role for PSK signaling in pollen, in vitro pollen tube (PT) growth was enhanced by exogenously added PSK while PTs of pskr1-3 pskr2-1 and of tpst-1 were shorter. In planta, growth of wild-type pollen in pskr1-3 pskr2-1 and tpst-1 flowers appeared slower than growth in wild-type flowers. But PTs did eventually reach the base of the style, suggesting that PT elongation rate may not be responsible for the reduced fertility. Detailed analysis of anthers, style and ovules did not reveal obvious developmental defects. By contrast, a high percentage of unfertilized ovules in pskr1-3 pskr2-1 and in tpst-1 siliques displayed loss of funicular PT guidance, suggesting that PSK signaling is required to guide the PT from the transmitting tract to the embryo sac. Cross-pollination experiments with wild-type, pskr1-3 pskr2-1 and tpst-1 male and female parents revealed that both the PT and the female sporophytic tissue and/or female gametophyte contribute to successful PT guidance via PSK signaling and to fertilization success.

Phytosulfokine control of growth occurs in the epidermis, is likely to be non-cell autonomous and is dependent on brassinosteroids.

Phytosulfokine (PSK) is a secreted disulfated pentapeptide that controls root and shoot growth. The ubiquitous expression of PSK precursor and of the LRR receptor kinase genes in Arabidopsis raised the question of whether PSK acts as an autocrine growth factor in planta. Expression of PSKR1 under the control of tissue- and cell type-specific promoters in a receptor null background strongly suggests that PSK is a non-cell autonomous signal that controls growth through localized activity in the epidermis. pskr1-3 pskr2-1 seedlings had shorter roots and hypocotyls than the wild type, whereas 35S: PSKR1 or 35S: PSKR2 seedlings were larger, indicating that receptor abundance limits growth in planta. The preferential expression of PSKR1 in the epidermis of CER6: PSKR1 pskr1-3 pskr2-1 seedlings was sufficient to promote wild-type growth. Moreover, in GL2:PSKR1 pskr1-3 pskr2-1 seedlings that express PSKR1 in atrichoblasts of the root epidermis, root growth was restored to wild-type levels. In pskr1-3 pskr2-1 seedlings, trichoblasts and atrichoblasts were shorter than in the wild type. Trichoblasts of GL2:PSKR1 pskr1-3 pskr2-1 seedlings, which are unable to sense PSK, nonetheless had acquired wild-type length, suggesting that PSK acts as a non-cell autonomous signal. Inhibition of brassinosteroid (BR) biosynthesis with brassinazole (BZ) caused a loss of responsiveness to PSK in wild-type, tpst-1 (tyrosylprotein sulfotransferase-1), PSKR1ox12 and CER6:PSKR1-3-1 seedlings, as did the genetic knock-out of BR synthesis in det2-1 and of BR perception in bri1-9, suggesting that BR mediates PSK-dependent growth. Quantitative PCR analysis of BR-related genes in wild-type, pskr1-3 pskr2-1, PSKR1ox and tpst-1 seedlings showed largely unchanged transcript levels of BR biosynthesis genes.