The MIXTA-LIKE transcription factor CsMYB6 regulates fruit spine and tubercule formation in cucumber

Highlights

• CsMYB6 is an important component of the molecular network that regulates fruit spine and tubercule formation in cucumber.

• CsMYB6 directly interacts with the key spine formation factor CsTTG1 to regulate spine development in cucumber.

• CsMYB6 negatively regulates tubercule formation in cucumber by interacting with the C2H2-type zinc-finger protein CsTu.

Abstract

Cucumber fruit wart composed of tubercule and spine (trichome on fruit) is not only an important fruit quality trait in cucumber production, but also a well-studied model for plant cell-fate determination. The development of spine is closely related to the initiation and formation of tubercule. The spine differentiation regulator CsGL1 has been proved to be epistatic to the tubercule initiation factor CsTu, which is the only connection to be identified between spine and tubercule formations. Our previous studies found that the MIXTA-LIKE transcription factor CsMYB6 can suppress fruit spine initiation, which is independent of CsGL1. How the formation of spine and tubercule is regulated at the molecular level by CsMYB6 remains poorly understood. In this study, we characterized cucumber 35S:CsMYB6 transgenic plants, which displayed an obvious reduction in the number and size of fruit spines and tubecules. Molecular analyses showed that CsMYB6 directly interacted with the key spine formation factor CsTTG1 in regulating the formation of fruit spine, and CsTu in regulating the initiation of fruit tubercule, respectively. Based on these evidences, a novel regulatory network is proposed by which CsMYB6/CsTTG1 and CsMYB6/CsTu complexes play an important role in regulating epidermal development, including spine formation and tubercule initiation in cucumber.

Introduction

Cucumber is one of the main facility cultivated vegetable in China, and is one of the most economically important vegetable crops in the world [1,2]. Cucumber fruit is usually covered with trichomes and tubercules, which are highly specialized structures originating from the epidermal cells [3]. Two distinct types of trichomes have been observed on cucumber fruits [[3], [4], [5]]. Type I, the glandular trichomes or bloom trichomes, produce fine white powdery secretions, which result in a coarse outer appearance cucumber fruits [4,5]. Type II, the spines, composed of a long trichome stalk and a spherical multicellular base, are much larger than the bloom trichomes [3,4]. Tubercules are usually located below the spines and derived from several layers of cells that lie near the spine base [2,6]. When spines are combined with tubercules, the cucumber fruits have a characteristic warty (Wty) trait, which is important for assessing fruit quality and thus directly affects the commercial value [2,6]. For example, the North American processing cucumbers (pickles) usually have sparse and large spines and tubercules on fruit surfaces, and cucumber fruits with dense and large spines and tubercules are more common in China fresh markets, while Mediterranean (beit alpha or mini) or European greenhouse cucumbers have no visible spines and tubercules on fruit surface, which make it easy for packing, transporting, cleaning, or keep less pesticide residues [2,6]. As such, there is considerable interest in understanding the regulatory mechanisms of fruit spines and tubercules development in cucumber in order to enhance breeding programs and the economic value of cucumber production.

Trichomes on leaves of Arabidopsis thaliana are unicellular, highly branched, and non-glandular structures. Numerous genes have been identified to regulate trichome initiation [[7], [8], [9], [10]]. The bHLH transcription factor GLABRA 3/ENHANCER OF GLABRA3 (AtGL3/AtEGL3) interacts with the MYB-like transcription factor GLABRA1 (AtGL1), and the WD-Repeat transcription factor TRANSPARENT TESTA GLABRA1 (AtTTG1), to form a trimeric complex, which activates trichome initiation by promoting the expression of HD-ZIP IV protein AtGL2 and AtEGL2 [8,10]. The negative regulatory factor AtTRY, AtCPC, AtETC1、AtETC2 and AtETC3 can compete with AtGL1 for interaction with AtGL3/AtEGL3 to suppress the initiation of trichome formation [11].

Cucumber fruit spines and tubercules are multicellular; however, only a few regulators controlling cucumber fruit spines and tubercules development are currently available and the molecular mechanism of cucumber spine and tubercules development is still poorly understood. The HD-ZIP I gene cucumber glabrous 1 (CsGL1) and HD-ZIP IV gene cucumber glabrous 3 (CsGL3) are essential for the formation of fruit spines in cucumber and genetic analyses showed that Csgl3 is epistatic to CsGL1 gene [5,[12], [13], [14], [15]]. Tuberculate fruit gene CsTu encoding a C₂H₂ zinc finger transcription factor was proved to be required for the tubercule formation in cucumber [2]. Genetic analyses showed that Csgl1 is epistatic to the CsTu, which is the only connection to be identified between spine and tubercule formations [2,5,15]. Moreover, we showed that the WD-Repeat protein CsTTG1 plays an important role in regulating fruit spines formation in cucumber, and unlike the function of its homologs in Arabidopsis, cotton, and many other species, the regulation of CsTTG1 in cucumber fruit spine is dosage-dependent [16].

MIXTA, derived from subgroup 9 of the R2R3-MYB family, was firstly characterized to affect shape of petals conical epidermal cells in Antirrhinum [17,18]. The MIXTA homologs in cotton, Arabidopsis and many other species have been identified to play positive or negative roles in regulating of trichome differentiation [7,[19], [20], [21], [22]]. GhMYB25 and GhMYB25-like, two MIXTA-like transcription factors in cotton, positively regulate fiber initiation and elongation [23,24]. Moreover, AtMYB106 and AtMYB16, two MIXTA-like transcription factors in Arabidopsis, negatively regulate trichome branch formation [25]. Our previous studies have shown that the MIXTA-like protein CsMYB6 regulates fruit spine initiation in cucumber [26]. Overexpression of CsMYB6 or CsTRY in cucumber results in fewer spines, while overexpression of CsMYB6 can inhibit rather than promote the expression of CsTRY [26]. Thus, the relationship between CsMYB6 and CsTRY seems complicated in cucumber, and how spine development regulated at the molecular level by CsMYB6 remains poorly understood. Moreover, CsMYB6 has been proved to act independently of CsGL1 in regulating the initiation of fruit spine [26]. In the current study, we characterized the cucumber 35S:CsMYB6 transgenic plants, and found that except for spine, the tubercule number was reduced on the fruit surface of 35S:CsMYB6 transgenic plants, indicating that CsMYB6 regulates tubercule formation through a molecular regulatory pathway which is independent of CsGL1. Using yeast two-hybrid assay and bimolecular fluorescence complementation assay, we found that CsMYB6 not only directly interacted with the key spine formation factor CsTTG1 to regulate the initiation of fruit spine, but also directly interacted with CsTu to regulate the initiation of fruit tubercule. These results indicate that CsMYB6 is an important component of the molecular network that regulates fruit spine and tubercule formation in cucumber. Our results also suggested a new connection between spine and tubercule formations.