Lazy1

[Giorgina Makara]

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TAC1 and LAZY1 are members of a gene family that regulates lateral shoot orientation in plants. TAC1 promotes outward orientations in response to light, while LAZY1 promotes upward shoot orientations in response to gravity via altered auxin transport. We performed genetic, molecular, and biochemical assays to investigate possible interactions between these genes. In Arabidopsis they were expressed in similar tissues and double mutants revealed the wide-angled lazy1 branch phenotype, indicating it is epistatic to the tac1 shoot phenotype. Surprisingly, the lack of TAC1 did not influence gravitropic shoot curvature responses. Combined, these results suggest TAC1 might negatively regulate LAZY1 to promote outward shoot orientations. However, additional results revealed that TAC1- and LAZY1 influence on shoot orientation is more complex than a simple direct negative regulatory pathway. Transcriptomes of Arabidopsis tac1 and lazy1 mutants compared to wild type under normal and gravistimulated conditions revealed few overlapping differentially expressed genes. Overexpression of each gene did not result in major branch angle differences. Shoot tip hormone levels were similar between tac1, lazy1, and Col, apart from exceptionally elevated levels of salicylic acid in lazy1. The data presented here provide a foundation for future study of TAC1 and LAZY1 regulation of shoot architecture.

An N-terminal transmembrane domain (TMD) and two C-terminal Nuclear Localization Signals (NLS) have been identified in LAZY1 and other LAZY proteins4,27. Yet, the importance or specific role of each domain may vary between species. Heterologously expressed GFP-tagged full-length and truncated LAZY1 proteins, along with in vitro assays, indicated monocot and dicot LAZY1 proteins, as well as Arabidopsis AtLAZY2, 3 and 4, associate with the plasma membrane and, in some cases, microtubules and nuclei4,5,20,28,29. The LAZY1 TMD was needed for membrane localization of transiently expressed rice and maize LAZY1-GFP in onion20,27. But an AtLAZY1 truncation lacking the TMD still localized to membranes in tobacco28. In addition, Arabidopsis protein fractionation assays suggested AtLAZY1 is a peripheral membrane protein, not a transmembrane one28. Lastly, the AtLAZY1 N-terminal Region I, upstream of the TMD was found to be important for both plasma membrane localization and branch angle control, as illustrated by site directed mutagenesis29. AtLAZY1 Regions II and V are also essential for LAZY1-directed branch angle control29. Interestingly, in Arabidopsis, nuclear localization is not needed for LAZY1-mediated branch angle control4. The lazy1 shoot phenotype was rescued by overexpressing a LAZY1 sequence containing a mutation in the NLS, which was shown to prevent nuclear localization in tobacco4.

LAZY1 transcription in rice is directly and positively regulated by the HEAT STRESS TRANSCRIPTION FACTOR 2D (HSF2D) protein upstream of auxin transport in response to gravistimulation31. Yeast-one-hybrid assays suggested LAZY4/DRO1 expression in wheat is directly regulated by AUXIN RESPONSE FACTOR 1 (ARF1) transcription factors11,31. In addition, yeast-two-hybrid and BiFC experiments determined that the maize LAZY1 (ZmLAZY1) protein bound to both a protein kinase (ZmPKC) which may be involved in PIN localization at the plasma membrane, and to a nuclear-localized Aux/IAA auxin signaling repressor protein (ZmIAA17)5. However, species-wide interpretations of results from maize should be cautioned because in addition to its role in gravitropism and shoot architecture, LAZY1 is also essential for tassel and ear development in maize5.

In contrast to the mechanistic knowledge about LAZY1, much less is known about TAC1. Its function in promoting wide lateral shoot angles is dosage dependent and light regulated3,13,14,15,16,17,18,19,32,33,34. TAC1 expression has been detected in shoot tissues in peach, poplar, and Arabidopsis, with higher expression detected in apical regions3,16,34. Further, expression in Arabidopsis was induced by light and eliminated after prolonged darkness or application of photosynthetic inhibitors18. Additionally, the narrow branch angle phenotype in Arabidopsis tac1 plants phenocopies the branch angles of wild type plants exposed to continuous shade or the chlorophyll biosynthesis inhibitor Norflurazon18. Lastly, AtTAC1 overexpression could not fully rescue the Arabidopsis mutant phenotype and it only partially prevented upright reorientation of branches in wild type plants in response to darkness3,18. These findings suggest additional pathways promote outward shoot orientations in Arabidopsis and/or the existence of post-transcriptional regulation of TAC1.

In all three tissue types, SA levels were substantially higher in lazy1 compared to both Col and tac1, with statistically significant increases in the primary shoot and branch tips (Fig. 5). Arabidopsis tac1 plants had less free IAA than Col in both shoot tips and stems, with the latter being statistically significant (Fig. 5A,B). In addition, lazy1 shoot tips had significantly greater IAA concentrations (Fig. 5A,B). No statistically significant differences in JA or ABA concentrations were detected between genotypes for the tissues we tested, although ABA levels in tac1 were slightly lower in stems and branch tips (Fig. 5).

After comparing expression profiles of each mutant to Col under the same conditions (normal or gravistimulated), a combined total of 829 differentially expressed genes (DEGs) were identified (Supplementary Data File D2). Fewer than 200 of these genes had expression changes greater than 2-fold, and very few DEGs had changes greater than 5-fold (Fig. 6A; Supplementary Data File D2). Most of the largest differences of expression were in response to gravistimulation. Interestingly, although no gravitropic bending differences between tac1 and Col were detected, in response to gravistimulation Col shoot tips had over two times the number of DEGS than tac1 (Fig. 6A, Supplementary Data File D2).

Although the precise nature of the relationship between these two genes remains unclear, the data presented here provide several important insights. First, relatively few genes were differentially expressed in tac1 and lazy1. Second, the majority of the DEGS were downregulated. Thus, if the LAZY1 protein can act as a repressor via its EAR motif, it likely targets few genes, and may repress one or multiple repressors. Also, potential transcriptional repression by LAZY1 in Arabidopsis is likely secondary to its gravitropism role, as nuclear localization was not required for maintaining branch orientation in this species4. Third, our RNAseq data are consistent with a role of LAZY1 in influencing gravitropic responses. Genes repressed in lazy1 plants included cell wall-related genes such as expansins and FLAs (whose expression was downregulated in lazy1), thereby promoting asymmetric cell elongation in shoots for positioning purposes. The lateral auxin transport genes PIN3, PIN4, and auxin influx gene AUX1 were also downregulated in lazy1.

In summary, the data described here highlight molecular and genetic aspects of TAC1- and LAZY1 function and their relationship to each other. Recent findings linking LAZY proteins to PIN3 localization via physical interactions with BRX-domain proteins suggest a possible mechanistic area where TAC1 and LAZY1 function could intersect30,45. One possibility is that TAC1 acts to integrate light perception into an ancient LAZY1-mediated gravity response pathway, thereby optimizing shoot positions for light capture. However, extensive experimentation is needed to build a comprehensive understanding of their independent and/or interdependent roles in regulating lateral shoot orientations in plants. The data provided here fills in some key knowledge gaps and provides new directions for future studies on TAC1 and LAZY1 branch angle control.

The authors thank Allison Brown, Erik Miller, Emma Acly, and Michaela Dunn at USDA-ARS Appalachian Fruit Research Station as well as Faith Hatt and Andrea Kohler at Michigan State university for help with miscellaneous plant care, genotyping, and/or data collection. We also would thank Drs. Takeshi Yoshihara and Edgar Spalding for kindly sharing promAtLAZY::GUS seed. Lastly, we are grateful to our anonymous reviewers, whose constructive feedback significantly improved this manuscript. This work was supported by Agriculture and Food Research Initiative Competitive grant 10891264 from the United States Department of Agriculture National Institute of Food and Agriculture, the National Science Foundation grant number 1339211, the United States Department of Agriculture National Institute of Food and Agriculture HATCH project 1013242, the Michigan State University Department of Horticulture, and Michigan State University AgBioResearch.

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We identified the gene responsible for three allelic lazy1 mutations of Japonica rice (Oryza sativa L.) by map-based cloning, complementation and RNA interference. Sequence analysis and database searches indicated that the wild-type gene (LAZY1) encodes a novel and unique protein (LAZY1) and that rice has no homologous gene. Two lazy1 mutants were LAZY1 null. Confirming and advancing the previously reported results on lazy1 mutants, we found the following. (i) Gravitropism is impaired, but only partially, in lazy1 coleoptiles. (ii) Circumnutation, observed in dark-grown coleoptiles, is totally absent from lazy1 coleoptiles. (iii) Primary roots of lazy1 mutants show normal gravitropism and circumnutation. (iv) LAZY1 is expressed in a tissue-specific manner in gravity-sensitive shoot tissues (i.e. coleoptiles, leaf sheath pulvini and lamina joints) and is little expressed in roots. (v) The gravitropic response of lazy1 coleoptiles is kinetically separable from that absent from lazy1 coleoptiles. (vi) Gravity-induced lateral translocation of auxin, found in wild-type coleoptiles, does not occur in lazy1 coleoptiles. Based on the genetic and physiological evidence obtained, it is concluded that LAZY1 is specifically involved in shoot gravitropism and that LAZY1-dependent and -independent signaling pathways occur in coleoptiles. It is further concluded that, in coleoptiles, only the LAZY1-dependent gravity signaling involves asymmetric distribution of auxin between the two lateral halves and is required for circumnutation.

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