Kazunari Nozue

Rhythmic growth explained by coincidence between internal and external cues

Most organisms use circadian oscillators to coordinate physiological and developmental processes such as growth with predictable daily environmental changes like sunrise and sunset. The importance of such coordination is highlighted by studies showing that circadian dysfunction causes reduced fitness in bacteria and plants, as well as sleep and psychological disorders in humans. Plant cell growth requires energy and water—factors that oscillate owing to diurnal environmental changes. Indeed, two important factors controlling stem growth are the internal circadian oscillator and external light levels. However, most circadian studies have been performed in constant conditions, precluding mechanistic study of interactions between the clock and diurnal variation in the environment. Studies of stem elongation in diurnal conditions have revealed complex growth patterns, but no mechanism has been described. Here we show that the growth phase of Arabidopsis seedlings in diurnal light conditions is shifted 8–12 h relative to plants in continuous light, and we describe a mechanism underlying this environmental response. We find that the clock regulates transcript levels of two basic helix–loop–helix genes, phytochrome-interacting factor 4 (PIF4) and PIF5, whereas light regulates their protein abundance. These genes function as positive growth regulators; the coincidence of high transcript levels (by the clock) and protein accumulation (in the dark) allows them to promote plant growth at the end of the night. Thus, these two genes integrate clock and light signalling, and their coordinated regulation explains the observed diurnal growth rhythms. This interaction may serve as a paradigm for understanding how endogenous and environmental signals cooperate to control other processes.

Genomic Analysis of Circadian Clock-, Light-, and Growth-Correlated Genes Reveals PHYTOCHROME-INTERACTING FACTOR5 as a Modulator of Auxin Signaling in Arabidopsis

Plants exhibit daily rhythms in their growth, providing an ideal system for the study of interactions between environmental stimuli such as light and internal regulators such as the circadian clock. We previously found that two basic loop-helix-loop transcription factors, PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, integrate light and circadian clock signaling to generate rhythmic plant growth in Arabidopsis (Arabidopsis thaliana). Here, we use expression profiling and real-time growth assays to identify growth regulatory networks downstream of PIF4 and PIF5. Genome-wide analysis of light-, clock-, or growth-correlated genes showed significant overlap between the transcriptomes of clock-, light-, and growth-related pathways. Overrepresentation analysis of growth-correlated genes predicted that the auxin and gibberellic acid (GA) hormone pathways both contribute to diurnal growth control. Indeed, lesions of GA biosynthesis genes retarded rhythmic growth. Surprisingly, GA-responsive genes are not enriched among genes regulated by PIF4 and PIF5, whereas auxin pathway and response genes are. Consistent with this finding, the auxin response is more severely affected than the GA response in pif4 pif5 double mutants and in PIF5-overexpressing lines. We conclude that at least two downstream modules participate in diurnal rhythmic hypocotyl growth: PIF4 and/or PIF5 modulation of auxin-related pathways and PIF-independent regulation of the GA pathway.

Protein abundances are more conserved than mRNA abundances across diverse taxa

Proteins play major roles in most biological processes; as a consequence, protein expression levels are highly regulated. While extensive post‐transcriptional, translational and protein degradation control clearly influence protein concentration and functionality, it is often thought that protein abundances are primarily determined by the abundances of the corresponding mRNAs. Hence surprisingly, a recent study showed that abundances of orthologous nematode and fly proteins correlate better than their corresponding mRNA abundances. We tested if this phenomenon is general by collecting and testing matching large‐scale protein and mRNA expression data sets from seven different species: two bacteria, yeast, nematode, fly, human, and rice. We find that steady‐state abundances of proteins show significantly higher correlation across these diverse phylogenetic taxa than the abundances of their corresponding mRNAs (p=0.0008, paired Wilcoxon). These data support the presence of strong selective pressure to maintain protein abundances during evolution, even when mRNA abundances diverge.

LeafJ: An ImageJ Plugin for Semi-automated Leaf Shape Measurement

High throughput phenotyping (phenomics) is a powerful tool for linking genes to their functions (see review and recent examples). Leaves are the primary photosynthetic organ, and their size and shape vary developmentally and environmentally within a plant. For these reasons studies on leaf morphology require measurement of multiple parameters from numerous leaves, which is best done by semi-automated phenomics tools. Canopy shade is an important environmental cue that affects plant architecture and life history; the suite of responses is collectively called the shade avoidance syndrome (SAS). Among SAS responses, shade induced leaf petiole elongation and changes in blade area are particularly useful as indices. To date, leaf shape programs (e.g. SHAPE, LAMINA, LeafAnalyzer, LEAFPROCESSOR) can measure leaf outlines and categorize leaf shapes, but can not output petiole length. Lack of large-scale measurement systems of leaf petioles has inhibited phenomics approaches to SAS research. In this paper, we describe a newly developed ImageJ plugin, called LeafJ, which can rapidly measure petiole length and leaf blade parameters of the model plant Arabidopsis thaliana. For the occasional leaf that required manual correction of the petiole/leaf blade boundary we used a touch-screen tablet. Further, leaf cell shape and leaf cell numbers are important determinants of leaf size. Separate from LeafJ we also present a protocol for using a touch-screen tablet for measuring cell shape, area, and size. Our leaf trait measurement system is not limited to shade-avoidance research and will accelerate leaf phenotyping of many mutants and screening plants by leaf phenotyping.

Shade Avoidance Components and Pathways in Adult Plants Revealed by Phenotypic Profiling

Shade from neighboring plants limits light for photosynthesis; as a consequence, plants have a variety of strategies to avoid canopy shade and compete with their neighbors for light. Collectively the response to foliar shade is called the shade avoidance syndrome (SAS). The SAS includes elongation of a variety of organs, acceleration of flowering time, and additional physiological responses, which are seen throughout the plant life cycle. However, current mechanistic knowledge is mainly limited to shade-induced elongation of seedlings. Here we use phenotypic profiling of seedling, leaf, and flowering time traits to untangle complex SAS networks. We used over-representation analysis (ORA) of shade-responsive genes, combined with previous annotation, to logically select 59 known and candidate novel mutants for phenotyping. Our analysis reveals shared and separate pathways for each shade avoidance response. In particular, auxin pathway components were required for shade avoidance responses in hypocotyl, petiole, and flowering time, whereas jasmonic acid pathway components were only required for petiole and flowering time responses. Our phenotypic profiling allowed discovery of seventeen novel shade avoidance mutants. Our results demonstrate that logical selection of mutants increased success of phenotypic profiling to dissect complex traits and discover novel components.

YUCCA auxin biosynthetic genes are required for Arabidopsis shade avoidance

Plants respond to neighbor shade by increasing stem and petiole elongation. Shade, sensed by phytochrome photoreceptors, causes stabilization of PHYTOCHROME INTERACTING FACTOR proteins and subsequent induction of YUCCA auxin biosynthetic genes. To investigate the role of YUCCA genes in phytochrome-mediated elongation, we examined auxin signaling kinetics after an end-of-day far-red (EOD-FR) light treatment, and found that an auxin responsive reporter is rapidly induced within 2 hours of far-red exposure. YUCCA2, 5, 8, and 9 are all induced with similar kinetics suggesting that they could act redundantly to control shade-mediated elongation. To test this hypothesis we constructed a yucca2, 5, 8, 9 quadruple mutant and found that the hypocotyl and petiole EOD-FR and shade avoidance responses are completely disrupted. This work shows that YUCCA auxin biosynthetic genes are essential for detectable shade avoidance and that YUCCA genes are important for petiole shade avoidance.

Quantitative Measurements of Xanthomonas Oryzae pv. Oryzae Distribution in Rice Using Fluorescent-Labeling

The rice host sensor, XA21, confers robust resistance to most strains of Xanthomonas oryzae pv. oryzae (Xoo), the casual agent of bacterial blight disease. Using in planta fluorescence imaging of Xoo strain PXO99Az expressing a green fluorescent protein (Xoo-gfp) we show that XA21 restricts Xoo spread at the point of infection. This noninvasive and quantitative method to measure spatial distribution of Xoo populations in planta facilitates detailed assessment of plant disease resistance.

Network analysis reveals a role for salicylic acid pathway components in shade avoidance

Shade avoidance involves complex regulation of multiple hormone network modules, and salicylic acid pathway genes are required for petiole shade avoidance. Plants have sophisticated mechanisms for sensing neighbor shade. To maximize their ability to compete for light, plants respond to shade through enhanced elongation and physiological changes. The shade avoidance response affects many different organs and growth stages, yet the signaling pathways underlying this response have mostly been studied in seedlings. We assayed transcriptome changes in response to shade across a 2-d time course in the wild type and 12 Arabidopsis (Arabidopsis thaliana) mutants. The resulting temporal map of transcriptional responses to shade defines early and late responses in adult plants, enabling us to determine connections between key signaling genes and downstream responses. We found a pervasive and unexpectedly strong connection between shade avoidance and genes related to salicylic acid, suggesting salicylic acid signaling to be an important shade avoidance growth regulator. We tested this connection and found that several mutants disrupting salicylic acid levels or signaling were defective in shade avoidance. The effect of these mutations on shade avoidance was specific to petiole elongation; neither hypocotyl nor flowering time responses were altered, thereby defining important stage-specific differences in the downstream shade avoidance signaling pathway. Shade treatment did not change salicylic acid levels, indicating that the mediation of shade avoidance by salicylic acid is not dependent on the modulation of salicylic acid levels. These results demonstrate that salicylic acid pathway genes also are key components of petiole shade avoidance.