The class II HD-ZIP JAIBA gene is involved in meristematic activity and important for gynoecium and fruit development in Arabidopsis

Abstract

Development and patterning of the gynoecium – and later the fruit – must be finely regulated to ensure the survival of the species that produces them. The process that leads to successful fruit formation starts at early stages of floral meristem development and follows a series of chronologically successive events. In a recent work we reported the functional characterization of the class II HD-ZIP JAIBA (JAB) gene. Mutant jab plants showed sporophytic defects in male and female reproductive development, and combined with the mutant crabs claw (crc) caused defects in the floral meristem (FM) determination process and gynoecium medial tissue development. Furthermore, the JAB protein interacted with transcription factors known to regulate meristematic activity, fruit development and FM determinacy. Preliminary results presented here suggest a genetic interaction between JAB and the gene SHOOT MERISTEMLESS (STM).

Keywords: :

• JAIBA
• class II HD-ZIP
• meristematic activity
• gynoecium
• indeterminacy

This article refers to:

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The indeterminate meristematic activity of plants is crucial for their development. However, complex determination programs are required in order to allow the differentiation and specialization of lateral organs. For example, flower development begins when the floral meristem (FM) is separated from the inflorescence meristem (IM). Then, the different floral organs start to develop in a predetermined sequence, giving rise to sepals, petals, stamens and finally the gynoecium. Gynoecium formation, the last step of floral organ initiation, starts at stage 6,^1,2 as the FM terminates, making the flower determinate with a fixed number of organs in each whorl.³

In plants, the gynoecium is one of the most complex organs. After fertilization, it develops into a fruit that has a very important function: to ensure the successful survival of the species that produces them. Proper fruit development depends on a series of events that must occur in a precise chronological order.

After the initiation of gynoecium development, the stem cell activity in the floral meristem must be carefully regulated so that the floral meristem determination process is performed correctly. This process seems to be an important event required for correct gynoecium development, and finally fruit development, because proper medial tissue development appears to be closely linked to the appropriate determination of the FM. Once the determination process has occurred, the next step is proper pollen and ovule development in the flower, followed by fertilization, which is necessary for seed production. In a recent article we showed evidence suggesting that the Arabidopsis class-II HD-ZIP JAIBA [‘x/h’aiba] (JAB) gene, together with CRABS CLAW (CRC), may perform a continuous modulation function of these series of events necessary to result in proper fruit development.⁴

In this work, we selected the gene JAB from a transcript profiling study during silique development.⁵ A T-DNA insertion line was identified and transgenic plants carrying the antisense construct (anti-JAB) were generated. First, phenotypic analysis of these lines showed that both the male and female gametes were affected. Most of the ovules have an empty embryo sac, while the pollen grains appear to be empty or deflated. This suggests that both gametes are affected during their biogenesis indicating that JAB may have an important role in these processes.

Second, a very interesting role of the JAB gene in the participation in floral meristem determination was revealed when the double mutant jab crc-1 was generated. In the double mutant the flowers showed both new phenotypic features not seen before in the single mutants and increased severity of previously observed phenotypes. These defects were classified into two categories: (1) defects in the floral meristem determination process; and (2) defects in the development of the gynoecium medial domain.

The floral meristem determination phenotypes (1) have been subdivided into two classes: class II that shows an increase of organ number before or during the gynoecium establishment (early effect); and class I that shows reiterations of floral organs inside the gynoecium (late effect).⁶ Interestingly, the jab crc-1 double mutant showed both classes in the same plant. This suggests that even when these two classes of phenotypes (production of extra organs) may result from distinct processes like an increased stem cell population or a prolonged maintenance of stem cells within the FM,³ there are genes such as JAB and CRC that may be involved throughout the sequential steps that sculpt a flower. It will be interesting to explore in more detail how the JAB gene is participating in the different steps of flower development.

In addition, the jab crc-1 double mutant gynoecia showed defects in medial domain development. When we analyzed the medial domain of single and double mutant gynoecia, we observed: first that the differentiation of the transmitting tract can occur independently of septum fusion; and second that the development of the medial tissue decreases when the indeterminacy phenotype increases. In gynoecia with a severe indeterminacy phenotype the septum virtually disappears, and only a few ovules develop. This last observation suggests that there is a connection between an adequate floral meristem determination process and correct gynoecium medial domain development. The discovery of new factors involved in these processes could help to confirm and understand this connection.

JAB encodes a transcription factor, and we analyzed its ability to form protein dimers to better understand in which molecular pathways JAB is involved. A uni-directional yeast two-hybrid (Y2H) assay was performed and the assay indicated that the JAB protein is able to form homodimers. Furthermore, JAB is able to form heterodimers with six different transcription factors, which are BREVIPEDICELLUS (BP), SHOOT MERISTEMLESS (STM), KNAT2 and PERIANTHIA (PAN); these proteins are associated with meristematic activity in different parts of the plant;^7-10 HECATE2 (HEC2) and STYLISH2 (STY2); involved in transmitting tract development, and stigma and style development; respectively.^11,12

The different single (jab) and double mutant (jab crc-1) phenotypes together with the Y2H assay results, and the fact that the expression pattern of these genes overlap in different plant tissues, suggest that JAB may be involved in processes such as inflorescence meristem regulation, floral meristem determination, gynoecium medial domain development and the development of style tissue through the formation of protein complexes with the proteins mentioned above.

Genetic Interaction Between JAIBA and SHOOT MERISTEMLESS

The protein-protein interaction between JAB and proteins encoded by genes involved in meristematic activity suggested that JAB also participates in this activity. In order to better understand the specific role of JAB in this process we decided to cross the jab mutant with the marker line STM::GUS.¹³ Preliminary results where the number of floral buds was taken as an indicator of inflorescence meristem activity suggested that JAB has a negative effect in this process.⁴

We analyzed the STM expression pattern in seedlings at 3 d post germination (dpg) in the jab and wild type backgrounds. The GUS signal was observed in the shoot apical meristem and surrounding areas in the wild type STM::GUS line. However, the GUS signal was significantly increased and expanded to the cotyledons in the jab mutant background (Fig. 1). This result suggests that in the jab mutant the STM gene is misexpressed and may therefore affect the normal meristematic activity. Whether the expression of STM is affected directly or indirectly by the JAB gene is an interesting question to be answered.

Figure 1.STM::GUS expression in 3-d-old seedlings. (A) GUS signal was detected in and around the SAM. (B) In the jab mutant the GUS signal was increased and expanded to the cotelydons. Scale bars in (A) and (B) 1 mm.

Conclusions

During their life cycle, plants require a continuous meristematic activity to maintain growth and to generate different types of organs. The fruit is one of the most complex organs produced by plants and a complex regulatory network is involved in its formation. For instance, meristematic activity needs to be controlled continuously in a very fine way. In the end, successful fruit development ensures survival of the species. Therefore, it can be expected that there are genes involved in this continuous regulation of this important developmental process. Different type of evidence suggests that the class-II HD-ZIP JAB gene, together with CRC is involved in this continuous regulation of fruit development to ensure its proper development.

Abbreviations:
JAB	=	JAIBA
CRC	=	CRABS CLAW
STM	=	SHOOT MERISTEMLESS
HD-ZIP	=	Homeodomain-leucine zipper
FM	=	floral meristem
IM	=	inflorescence meristem
Y2H	=	yeast two-hybrid

Acknowledgments

We would like to thank Mitsuhiro Aida for the STM::GUS line. VZM was supported by the Mexican National Council of Science and Technology (CONACyT) fellowship (210100). This work was financed by the CONACyT grant 82826 and CONCyTEG grant 08–03-K662–116.