Phosphatidylinositol‐specific phospholipase C2 functions in auxin‐modulated root development

Nine phosphatidylinositol‐specific phospholipases C (PLCs) have been identified in the Arabidopsis genome; among the importance of PLC2 in reproductive development is significant. However, the role of PLC2 in vegetative development such as in root growth is elusive. Here, we report that plc2 mutants displayed multiple auxin‐defective phenotypes in root development, including short primary root, impaired root gravitropism, and inhibited root hair growth. The DR5:GUS expression and the endogenous indole‐3‐acetic acid (IAA) content, as well as the responses of a set of auxin‐related genes to exogenous IAA treatment, were all decreased in plc2 seedlings, suggesting the influence of PLC2 on auxin accumulation and signalling. The root elongation of plc2 mutants was less sensitive to the high concentration of exogenous auxins, and the application of 1‐naphthaleneacetic acid or the auxin transport inhibitor N‐1‐naphthylphthalamic acid could rescue the root hair growth of plc2 mutants. In addition, the PIN2 polarity and cycling in plc2 root epidermis cells were altered. These results demonstrate a critical role of PLC2 in auxin‐mediated root development in Arabidopsis, in which PLC2 influences the polar distribution of PIN2.     

Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid‐mediated defenses

Auxin is a key plant growth regulator that also impacts plant–pathogen interactions. Several lines of evidence suggest that the bacterial plant pathogen Pseudomonas syringae manipulates auxin physiology in Arabidopsis thaliana to promote pathogenesis. Pseudomonas syringae strategies to alter host auxin biology include synthesis of the auxin indole‐3‐acetic acid (IAA) and production of virulence factors that alter auxin responses in host cells. The application of exogenous auxin enhances disease caused by P. syringae strain DC3000. This is hypothesized to result from antagonism between auxin and salicylic acid (SA), a major regulator of plant defenses, but this hypothesis has not been tested in the context of infected plants. We further investigated the role of auxin during pathogenesis by examining the interaction of auxin and SA in the context of infection in plants with elevated endogenous levels of auxin. We demonstrated that elevated IAA biosynthesis in transgenic plants overexpressing the YUCCA 1 (YUC1) auxin biosynthesis gene led to enhanced susceptibility to DC3000. Elevated IAA levels did not interfere significantly with host defenses, as effector‐triggered immunity was active in YUC1‐overexpressing plants, and we observed only minor effects on SA levels and SA‐mediated responses. Furthermore, a plant line carrying both the YUC1‐overexpression transgene and the salicylic acid induction deficient 2 (sid2) mutation, which impairs SA synthesis, exhibited additive effects of enhanced susceptibility from both elevated auxin levels and impaired SA‐mediated defenses. Thus, in IAA overproducing plants, the promotion of pathogen growth occurs independently of suppression of SA‐mediated defenses.     

CsSPL functions as an adaptor between HD‐ZIP III and CsWUS transcription factors regulating anther and ovule development in Cucumis sativus (cucumber)

Anther and ovule genesis preconditions crop fertilization and fruit production; however, coordinative regulation of anther and ovule development and underlying molecular pathways remain largely elusive. Here, we found that SPOROCYTELESS (SPL)/NOZZLE (NZZ) expression was nearly abolished in a Cucumis sativus (cucumber) mutant with severely defective anther and ovule development. CsSPL was expressed specifically in the developing anthers and ovules. Knock‐down of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development. Importantly, CsSPL directly interacted with CsWUS (WUSCHEL) in the nucellus and YABBY family genes in integuments, and positively regulated CsWUS expression, meanwhile the HD‐ZIP III gene CsPHB (PHABULOSA), expressed specifically in the nucellus, promoted CsSPL expression by binding to the CsSPL promoter. Thus, CsSPL acts as an adaptor to link CsPHB and CsWUS functioning, underpinning a previously unidentified regulatory pathway orchestrating sex organ development in planta. In addition, auxin accumulation was reduced in the reproductive organs of CsSPL knock‐down plants. Biochemical analyses further showed that CsSPL stimulated the expression of AUXIN RESPONSE FACTOR 3 (CsARF3), and was positively regulated by CsARF13 during reproductive organ development, indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.     

Arabidopsis DOK1 encodes a functional dolichol kinase involved in reproduction

Dolichol phosphate (Dol‐P) serves as a carrier of complex polysaccharides during protein glycosylation. Dol‐P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol‐PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome‐wide search for cytidylyltransferase motif‐containing proteins in Arabidopsis, and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N‐linked glycosylation of the S. cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T‐DNA‐tagged AtDOK1 mutants, dok1‐1 and dok1‐2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis.     

Using RAD‐seq to develop sex‐linked markers in a haplodiplontic alga

For many taxa, including isomorphic haplodiplontic macroalgae, determining sex and ploidy is challenging, thereby limiting the scope of some population demographic and genetic studies. Here, we used double‐digest restriction site‐associated DNA sequencing (ddRAD‐seq) to identify sex‐linked molecular markers in the widespread red alga Agarophyton vermiculophyllum. In the ddRAD‐seq library, we included 10 female gametophytes, 10 male gametophytes, and 16 tetrasporophytes from one native and one non‐native site (N = 40 gametophytes and N = 32 tetrasporophytes total). We identified seven putatively female‐linked and 19 putatively male‐linked sequences. Four female‐ and eight male‐linked markers amplified in all three life cycle stages. Using one female‐ and one male‐linked marker that were sex‐specific, we developed a duplex PCR and tested the efficacy of this assay on a subset of thalli sampled at two sites in the non‐native range. We confirmed ploidy based on the visual observation of reproductive structures and previous microsatellite genotyping at 10 polymorphic loci. For 32 vegetative thalli, we were able to assign sex and confirm ploidy in these previously genotyped thalli. These markers will be integral to ongoing studies of A. vermiculophyllum invasion. We discuss the utility of RAD‐seq over other approaches previously used, such as RAPDs (random amplified polymorphic DNA), for future work designing sex‐linked markers in other haplodiplontic macroalgae for which genomes are lacking.     

Auxin biosynthetic gene TAR2 is involved in low nitrogen‐mediated reprogramming of root architecture in Arabidopsis

In plants, the plasticity of root architecture in response to nitrogen availability largely determines nitrogen acquisition efficiency. One poorly understood root growth response to low nitrogen availability is an observed increase in the number and length of lateral roots (LRs). Here, we show that low nitrogen‐induced Arabidopsis LR growth depends on the function of the auxin biosynthesis gene TAR2 (tryptophan aminotransferase related 2). TAR2 was expressed in the pericycle and the vasculature of the mature root zone near the root tip, and was induced under low nitrogen conditions. In wild type plants, low nitrogen stimulated auxin accumulation in the non‐emerged LR primordia with more than three cell layers and LR emergence. Conversely, these low nitrogen‐mediated auxin accumulation and root growth responses were impaired in the tar2‐c null mutant. Overexpression of TAR2 increased LR numbers under both high and low nitrogen conditions. Our results suggested that TAR2 is required for reprogramming root architecture in response to low nitrogen conditions. This finding suggests a new strategy for improving nitrogen use efficiency through the engineering of TAR2 expression in roots.     

The DC1‐domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain‐containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine‐ and histidine‐rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre‐vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 – a member of the SNARE vesicle‐associated protein family and previously related to a sterol‐binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.     

Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

microRNA159‐targeted SlGAMYB transcription factors are required for fruit set in tomato

The transition from flowering to fruit production, namely fruit set, is crucial to ensure successful sexual plant reproduction. Although studies have described the importance of hormones (i.e. auxin and gibberellins) in controlling fruit set after pollination and fertilization, the role of microRNA‐based regulation during ovary development and fruit set is still poorly understood. Here we show that the microRNA159/GAMYB1 and ‐2 pathway (the miR159/GAMYB1/2 module) is crucial for tomato ovule development and fruit set. MiR159 and SlGAMYBs were expressed in preanthesis ovaries, mainly in meristematic tissues, including developing ovules. SlMIR159‐overexpressing tomato cv. Micro‐Tom plants exhibited precocious fruit initiation and obligatory parthenocarpy, without modifying fruit shape. Histological analysis showed abnormal ovule development in such plants, which led to the formation of seedless fruits. SlGAMYB1/2 silencing in SlMIR159‐overexpressing plants resulted in misregulation of pathways associated with ovule and female gametophyte development and auxin signalling, including AINTEGUMENTA‐like genes and the miR167/SlARF8a module. Similarly to SlMIR159‐overexpressing plants, SlGAMYB1 was downregulated in ovaries of parthenocarpic mutants with altered responses to gibberellins and auxin. SlGAMYBs likely contribute to fruit initiation by modulating auxin and gibberellin responses, rather than their levels, during ovule and ovary development. Altogether, our results unveil a novel function for the miR159‐targeted SlGAMYBs in regulating an agronomically important trait, namely fruit set.     

CLUMSY VEIN,the Arabidopsis DEAH‐box Prp16 ortholog,is required for auxin‐mediated development

Pre‐messenger RNA (pre‐mRNA) splicing is essential in eukaryotic cells. In animals and yeasts, the DEAH‐box RNA‐dependent ATPase Prp16 mediates conformational change of the spliceosome, thereby facilitating pre‐mRNA splicing. In yeasts, Prp16 also plays an important role in splicing fidelity. Conversely, PRP16 orthologs in Chlamydomonas reinhardtii and nematode do not have an important role in general pre‐mRNA splicing, but are required for gene silencing and sex determination, respectively. Functions of PRP16 orthologs in higher plants have not been described until now. Here we show that the CLUMSY VEIN (CUV) gene encoding the unique Prp16 ortholog in Arabidopsis thaliana facilitates auxin‐mediated development including male‐gametophyte transmission, apical–basal patterning of embryonic and gynoecium development, stamen development, phyllotactic flower positioning, and vascular development. cuv‐1 mutation differentially affects splicing and expression of genes involved in auxin biosynthesis, polar auxin transport, auxin perception and auxin signaling. The cuv‐1 mutation does not have an equal influence on pre‐mRNA substrates. We propose that Arabidopsis PRP16/CUV differentially facilitates expression of genes, which include genes involved in auxin biosynthesis, transport, perception and signaling, thereby collectively influencing auxin‐mediated development.     

Segregation distortion in Arabidopsis <Emphasis Type="Italic">gametophytic factor 1 </Emphasis>(<Emphasis Type="Italic">gfa1</Emphasis>) mutants is caused by a deficiency of an essential RNA splicing factor

The female and male gametophytes are critical components of the angiosperm life cycle and are essential for the reproductive process. The gametophytes share many essential cellular processes with each other and with the sporophyte generation. As a consequence, these processes can only be analyzed genetically in the gametophyte generation. Here, we report the characterization of the gametophytic factor 1 (gfa1) mutant. The gfa1 mutation exhibits reduced transmission through both the female and male gametophytes. Reduced transmission through the female gametophyte is due to an effect on female gametophyte development. By contrast, development of the pollen grain is not affected in gfa1; rather, reduced transmission is likely due to an effect on pollen tube growth. We have identified multiple T-DNA-insertion alleles of gfa1 in a gene encoding a protein with high similarity to Snu114/U5-116 kD proteins from yeast and animals required for normal pre-mRNA splicing. Consistent with its predicted function, the GFA1 gene (At1g06220) is expressed throughout the plant. Together, these data suggest that GFA1 functions in mRNA splicing during the plant life cycle. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.