Auxin receptors as integrators of developmental and hormonal signals during reproductive development in pea.

Auxins regulate many aspects of plant growth and development. In pea, three of the five TIR1/AFB members (PsTIR1a, PsTIR1b, and PsAFB2) have been implicated in auxin-related responses during fruit/seed development; however, the roles of PsAFB4 and PsAFB6 in these processes are unknown. Using yeast two-hybrid assays, we found that all five pea TIR1/AFB receptor proteins interacted with the pea AUX/IAAs PsIAA6 and/or PsIAA7 in an auxin-dependent manner, a requirement for functional auxin receptors. All five auxin receptors are expressed in young ovaries (pericarps) and rapidly developing seeds, with overlapping and unique developmental and hormone-regulated gene expression patterns. Pericarp PsAFB6 expression was suppressed by seeds and increased in response to deseeding, and exogenous hormone treatments suggest that seed-derived auxin and deseeding-induced ethylene are involved in these responses, respectively. Ethylene-induced elevation of pericarp PsAFB6 expression was associated with 4-Cl-IAA-specific reduction in ethylene responsiveness. In developing seeds, expression of PsTAR2 and PsYUC10 auxin biosynthesis genes was associated with high auxin levels in seed coat and cotyledon tissues, and PsAFB2 dominated the seed tissue transcript pool. Overall, auxin receptors had overlapping and unique developmental and hormone-regulated gene expression patterns during fruit/seed development, suggesting mediation of diverse responses to auxin, with PsAFB6 linking auxin and ethylene signaling.

TIR1 auxin receptors are implicated in the differential response to 4-Cl-IAA and IAA in developing pea fruit.

The auxins indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA) occur naturally in pea (Pisum sativum); however, only 4-Cl-IAA mimics the presence of seeds in stimulating pericarp growth. To examine if this differential auxin effect is mediated through TIR1/AFB auxin receptors, pea TIR1 and AFB2 homologs were functionally characterized in Arabidopsis, and receptor expression, and auxin distribution and action were profiled in developing pea fruits. PsTIR1a, PsTIR1b, and PsAFB2 restored the auxin-sensitive root growth response to the mutant Arabidopsis seedlings Attir1-10 and/or Attir1-10 afb2-3. Expression of PsTIR1 or AtTIR1 in Attir1-10 afb2-3 mutants also restored the greater root inhibitory response of 4-Cl-IAA compared to that of IAA, implicating TIR1 receptors in this response. The ability of 4-Cl-IAA to stimulate a stronger DR5::GUS auxin response than IAA at the same concentration in pea pericarps was associated with its ability to enrich the auxin-receptor transcript pool with PsTIR1a and PsAFB2 by decreasing the transcript abundance of PsTIR1b (mimicking results in pericarps with developing seeds). Therefore, the markedly different effect of IAA and 4-Cl-IAA on pea fruit growth may at least partially involve TIR1/AFB receptors and the differential modulation of their population, resulting in specific Aux/IAA protein degradation that leads to an auxin-specific tissue response.

Heat stress differentially modifies ethylene biosynthesis and signaling in pea floral and fruit tissues.

KEY MESSAGE: Ethylene biosynthesis is regulated in reproductive tissues in response to heat stress in a manner to optimize resource allocation to pollinated fruits with developing seeds. High temperatures during reproductive development are particularly detrimental to crop fruit/seed production. Ethylene plays vital roles in plant development and abiotic stress responses; however, little is known about ethylene's role in reproductive tissues during development under heat stress. We assessed ethylene biosynthesis and signaling regulation within the reproductive and associated tissues of pea during the developmental phase that sets the stage for fruit-set and seed development under normal and heat-stress conditions. The transcript abundance profiles of PsACS [encode enzymes that convert S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC)] and PsACO (encode enzymes that convert ACC to ethylene), and ethylene evolution were developmentally, environmentally, and tissue-specifically regulated in the floral/fruit/pedicel tissues of pea. Higher transcript abundance of PsACS and PsACO in the ovaries, and PsACO in the pedicels was correlated with higher ethylene evolution and ovary senescence and pedicel abscission in fruits that were not pollinated under control temperature conditions. Under heat-stress conditions, up-regulation of ethylene biosynthesis gene expression in pre-pollinated ovaries was also associated with higher ethylene evolution and lower retention of these fruits. Following successful pollination and ovule fertilization, heat-stress modified PsACS and PsACO transcript profiles in a manner that suppressed ovary ethylene evolution. The normal ethylene burst in the stigma/style and petals following pollination was also suppressed by heat-stress. Transcript abundance profiles of ethylene receptor and signaling-related genes acted as qualitative markers of tissue ethylene signaling events. These data support the hypothesis that ethylene biosynthesis is regulated in reproductive tissues in response to heat stress to modulate resource allocation dynamics.

Regulation of ethylene-related gene expression by indole-3-acetic acid and 4-chloroindole-3-acetic acid in relation to pea fruit and seed development.

In pea, the auxins 4-chloroindole-3-acetic acid (4-Cl-IAA) and indole-3-acetic acid (IAA) occur naturally; however, only 4-Cl-IAA stimulates pericarp growth and gibberellin (GA) biosynthesis, and inhibits the ethylene response in deseeded ovaries (pericarps), mimicking the presence of seeds. Expression of ovary ethylene biosynthesis genes was regulated similarly in most cases by the presence of 4-Cl-IAA or seeds. PsACS1 [which encodes an enzyme that synthesizes 1-aminocyclopropane-1-carboxylic acid (ACC)] transcript abundance was high in pericarp tissue adjacent to developing seeds following pollination. ACC accumulation in 4-Cl-IAA-treated deseeded pericarps was driven by high PsASC1 expression (1800-fold). 4-Cl-IAA, but not IAA, also suppressed the pericarp transcript levels of PsACS4. 4-Cl-IAA increased PsACO1 and decreased PsACO2 and PsACO3 expression (enzymes that convert ACC to ethylene) but did not change ACO enzyme activity. Increased ethylene was countered by a 4-Cl-IAA-specific decrease in ethylene responsiveness potentially via modulation of pericarp ethylene receptor and signaling gene expression. This pattern did not occur in IAA-treated pericarps. Overall, the effect of 4-Cl-IAA and IAA on ethylene biosynthesis gene expression generally explains the ethylene evolution patterns, and their effects on GA biosynthesis and ethylene signaling gene expression explain the tissue response patterns in young pea ovaries.

Impact of Susceptibility on Plant Hormonal Composition during Clubroot Disease Development in Canola (Brassica napus).

Clubroot, caused by Plasmodiophora brassicae, is a soilborne disease of crucifers associated with the formation of large root galls. This root enlargement suggests modulation of plant hormonal networks by the pathogen, stimulating cell division and elongation and influencing host defense. We studied physiological changes in two Brassica napus cultivars, including plant hormone profiles-salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA), the auxin indole-3-acetic acid (IAA), and the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC)-along with their selected derivatives following inoculation with virulent and avirulent P. brassicae pathotypes. In susceptible plants, water uptake declined from the initial appearance of root galls by 21 days after inoculation, but did not have a significant effect on photosynthetic rate, stomatal conductance, or leaf chlorophyll levels. Nonetheless, a strong increase in ABA levels indicated that hormonal mechanisms were triggered to cope with water stress due to the declining water uptake. The free SA level in the roots increased strongly in resistant interactions, compared with a relatively minor increase during susceptible interactions. The ratio of conjugated SA to free SA was higher in susceptible interactions, indicating that resistant interactions are linked to the plant's ability to maintain higher levels of bioactive free SA. In contrast, JA and its biologically active form JA-Ile declined up to 7-fold in susceptible interactions, while they were maintained during resistant interactions. The ACC level increased in the roots of inoculated plants by 21 days, irrespective of clubroot susceptibility, indicating a role of ethylene in response to pathogen interactions that is independent of disease severity. IAA levels at early and later infection stages were lower only in susceptible plants, suggesting a modulation of auxin homeostasis by the pathogen relative to the host defense system.

Comparative Transcriptomic Analysis of Gene Expression Inheritance Patterns Associated with Cabbage Head Heterosis.

The molecular mechanism of heterosis or hybrid vigor, where F1 hybrids of genetically diverse parents show superior traits compared to their parents, is not well understood. Here, we studied the molecular regulation of heterosis in four F1 cabbage hybrids that showed heterosis for several horticultural traits, including head size and weight. To examine the molecular mechanisms, we performed a global transcriptome profiling in the hybrids and their parents by RNA sequencing. The proportion of genetic variations detected as single nucleotide polymorphisms and small insertion-deletions as well as the numbers of differentially expressed genes indicated a larger role of the female parent than the male parent in the genetic divergence of the hybrids. More than 86% of hybrid gene expressions were non-additive. More than 81% of the genes showing divergent expressions showed dominant inheritance, and more than 56% of these exhibited maternal expression dominance. Gene expression regulation by cis-regulatory mechanisms appears to mediate most of the gene expression divergence in the hybrids; however, trans-regulatory factors appear to have a higher effect compared to cis-regulatory factors on parental expression divergence. These observations bring new insights into the molecular mechanisms of heterosis during the cabbage head development.