Identification, expression, and functional analysis of CLE genes in radish (Raphanus sativus L.) storage root

Abstract

Background: Radish (Raphanus sativus L.) is a widespread agricultural plant forming storage root due to extensive secondary growth which involves cambium proliferation and differentiation of secondary conductive tissues. Closely related to the model object Arabidopsis thaliana, radish is a suitable model for studying processes of secondary growth and storage root development. CLE peptides are a group of peptide phytohormones which play important role in the regulation of primary meristems such as SAM, RAM, and procambium, as well as secondary meristems. However, the role of CLE peptides in lateral growth of root during storage root formation has not been studied to date.

Results: In present work we studied the role of CLE peptides in the development of storage root in radish. We have identified 18 CLE genes of radish (RsCLEs) and measured their expression in various plant organs and also at different stages of root development in R. sativus and Raphanus raphanistrum-its close relative which does not form storage root. We observed significant decline of expression levels for genes RsCLE1, 2, 11, 13, and 16, and also multifold increase of expression levels for genes RsCLE19, and 41 during secondary root growth in R. sativus but not in R. raphanistrum. Expression of RsCLE 2, 19, and 41 in R. sativus root was confined to certain types of tissues while RsCLE1, 11, 13, and 16 expressed throughout the root. Experiments on overexpression of RsCLE2, 19 and 41 or treatment of radish plants with synthetic CLE peptides revealed that CLE19 and CLE2 increase the number of xylem elements, and CLE41 induces the formation of extra cambium foci in secondary xylem. Expression levels of RsCLE2 and 19 strongly decrease in response to exogenous cytokinin, while auxin causes dramatic increase of RsCLE19 expression level and decrease of RsCLE41 expression.

Conclusions: Our data allow us to hypothesize about the role of RsCLE2, 19 and 41 genes in the development of storage root of Raphanus sativus, e.g. RsCLE19 may play a role in auxin-dependent processes of xylem differentiation and RsCLE41 stimulates cambium activity.

Fig. 1

Comparison of the anatomical structure of Raphanus raphanistrum (a) and Raphanus sativus (b) roots (30 day old plants). xyI—primary xylem, xyII—secondary xylem, ca—cambium, ph—phloem, co—cortex, vs—vessels, mxp—mechanical xylem parenchyma, sxp—storage xylem parenchyma, rr—radial ray

Fig. 2

qRT-PCR analysis of RsCLE genes expression. a, b RsCLE genes expression in Raphanus raphanistrum (a) and Raphanus sativus (b) roots (line 27) at different stage of development. Expression levels are shown relative to the expression found in the root of 7-day old seedlings. c RsCLE genes expression in different tissues of Raphanus sativus storage root. Error bars indicate standard deviation of three technical repeats. (P < 0.05 - *, P < 0.01 - **, P < 0.001 - ***)

Fig. 3

Effect of CLE-peptides on the development of Raphanus raphanistrum and Raphanus sativus roots. a-h—Effect of exogenous CLE peptides supplying. Transverse sections of Raphanus sativus line 19 (a-d) and Raphanus raphanistrum (e-h) roots of 15-day old plants after 7 days cultivation of medium with synthetic CLE-peptides: control (CLE41def) (a, e), CLE19p (b, f), CLE41p (c, g), and CLE2p (d, h). i-m Effect of RsCLE41 overexpression on development of meristematic foci in the central part of xylem zone. i, j transverse sections of mature (30 day old) roots of Raphanus sativus line 19: i—GUS overexpression (control); j RsCLE41 overexpression. k, l—meristematic foci in the central part of xylem zone of roots with GUS overexpression (k) and RsCLE41 overexpression (l). m number of xylem elements on transverse section of Raphanus sativus root of 250 μM in diameter (P < 0.01 - **, P < 0.001 - ***). n qRT-PCR analysis of RsCLE41 expression in GUS- overexpressing and RsCLE41-overexpressing Raphanus sativus roots (lines 19 and 27)

Fig. 4

Effect of exogenous cytokinin (BAP, 10 mM) (a) and auxin (NAA, 10 mM) (b) on expression of RsCLE2, 19 and 41 genes in upper part of root of Raphanus sativus seedlings. Error bars indicate standard deviation of three technical repeats. (P < 0.05 - *, P < 0.01 - **, P < 0.001 - ***)